Nanocrystals, compositions, and methods that aid particle transport in mucus

ABSTRACT

Nanocrystals, compositions, and methods that aid particle transport in mucus are provided. In some embodiments, the compositions and methods involve making mucus-penetrating particles (MPP) without any polymeric carriers, or with minimal use of polymeric carriers. The compositions and methods may include, in some embodiments, modifying the surface coatings of particles formed of pharmaceutical agents that have a low water solubility. Such methods and compositions can be used to achieve efficient transport of particles of pharmaceutical agents though mucus barriers in the body for a wide spectrum of applications, including drug delivery, imaging, and diagnostic applications. In certain embodiments, a pharmaceutical composition including such particles is well-suited for administration routes involving the particles passing through a mucosal barrier.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/616,799 filed Jun. 7, 2017, which is a continuation of U.S.patent application Ser. No. 15/354,704 filed Nov. 17, 2016, now U.S.Pat. No. 9,737,491, which is a continuation of U.S. patent applicationSer. No. 15/187,552 filed Jun. 20, 2016, now U.S. Pat. No. 9,532,955,which is a continuation of U.S. patent application Ser. No. 14/731,921filed Jun. 5, 2015, now U.S. Pat. No. 9,393,212, which is a continuationof Ser. No. 13/886,493, filed May 3, 2013, now U.S. Pat. No. 9,056,057,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application No. 61/642,227, filed May 3, 2012 and entitled“Nanocrystals, Compositions, and Methods That Aid Particle Transport inMucus”, all of which are incorporated herein by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.R33AI079740, R33AI094519, R01HD062844, and R01CA140746 awarded by theNational Institutes of Heath. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention generally relates to nanocrystals, compositions,and methods that aid particle transport in mucus.

BACKGROUND OF THE INVENTION

A mucus layer present at various points of entry into the body,including the eyes, nose, lungs, gastrointestinal tract, and femalereproductive tract, is naturally adhesive and serves to protect the bodyagainst pathogens, allergens, and debris by effectively trapping andquickly removing them via mucus turnover. For effective delivery oftherapeutic, diagnostic, or imaging particles via mucus membranes, theparticles must be able to readily penetrate the mucus layer to avoidmucus adhesion and rapid mucus clearance. Several lines of evidencesuggest that conventional nanoparticles are not capable of crossingmucosal barriers. However, it has been recently demonstrated thatpolymeric nanoparticles (degradable or not) modified with a specialsurface coating (covalently or non-covalently) can diffuse inphysiologically think mucus samples nearly as rapidly as they would inwater. Such polymer-based mucus-penetrating particles (MPP) canencapsulate various therapeutic, imaging, or diagnostic agents to enabledrug delivery, diagnostic, or imaging applications.

Nevertheless, polymer-based MPP may have several inherent limitationscompared to unencapsulated particles of pharmaceutical agents. Inparticular, in light of drug delivery applications these limitations mayinclude: 1) Inherently lower drug loading; 2) Less convenient dosageform, as reconstitution from a dry powder storage form may be requiredfor polymeric nanoparticles; 3) Potentially increased toxicity; 4)Chemical and physical stability concerns; and 5) Increased manufacturingcomplexity. Accordingly, improvements in compositions and methodsinvolving mucus-penetrating particles for delivery of pharmaceuticalagents would be beneficial.

SUMMARY OF THE INVENTION

The present description generally relates to nanocrystals, compositions,and methods that aid particle transport in mucus. In some embodiments,the compositions and methods involve mucus-penetrating particles withoutany polymeric carriers, or with minimal use of polymeric carriers. Thesubject matter of this application involves, in some cases, interrelatedproducts, alternative solutions to a particular problem, and/or aplurality of different uses of structures and compositions.

In one set of embodiments, a method of forming coated particles isprovided. The method involves combining core particles with a solutioncomprising a surface-altering agent, wherein the core particles comprisea solid pharmaceutical agent or a salt thereof, wherein the agent orsalt has a solubility of less than or equal to about 1 mg/mL in thesolution at 25° C., and wherein the pharmaceutical agent or salt thereofconstitutes at least about 80 wt % of each of the core particles. Themethod also involves coating the core particles with thesurface-altering agent to form coated particles, wherein thesurface-altering agent comprises a triblock copolymer comprising ahydrophilic block—hydrophobic block—hydrophilic block configuration,wherein the hydrophobic block has a molecular weight of at least about 2kDa, and the hydrophilic blocks constitute at least about 15 wt % of thetriblock copolymer, wherein the hydrophobic block associates with thesurface of the core particles, wherein the hydrophilic block is presentat the surface of the coated particles and renders the coated particleshydrophilic, and wherein the coated particles have a relative velocityof greater than 0.5 in mucus.

In another set of embodiments, a composition comprising a plurality ofcoated particles is provided. The coated particle comprises a coreparticle comprising a solid pharmaceutical agent or a salt thereof,wherein the agent or salt has an aqueous solubility of less than orequal to about 1 mg/mL at 25° C. at any point throughout the pH range,wherein the pharmaceutical agent or salt thereof constitutes at leastabout 80 wt % of the core particle. The coated particle also includes acoating comprising a surface-altering agent surrounding the coreparticle, wherein the surface-altering agent comprises a triblockcopolymer comprising a hydrophilic block—hydrophobic block—hydrophilicblock configuration, wherein the hydrophobic block has a molecularweight of at least about 2 kDa, and the hydrophilic blocks constitute atleast about 15 wt % of the triblock copolymer, wherein the hydrophobicblock associates with the surface of the core particle, wherein thehydrophilic block is present at the surface of the coated particle andrenders the coated particle hydrophilic, and wherein thesurface-altering agent is present on the surface of the core particle ata density of at least about 0.001 molecules per nanometer squared. Thecoated particles have a relative velocity of greater than 0.5 in mucus.

In another set of embodiments, a method of forming coated comprisesproviding a pharmaceutical agent and precipitating the pharmaceuticalagent by forming a salt in an aqueous solution in the presence of asurface-altering agent to form core particles of the pharmaceuticalagent, wherein the salt has a lower aqueous solubility than thepharmaceutical agent in the non-salt form, the aqueous solubility of thesalt being less than about 1 mg/mL at 25° C. at any point throughout thepH range, and wherein the surface-altering agent is present at aconcentration of at least about 0.01% (w/v) in the aqueous solution. Themethod involves coating the core particles with the surface-alteringagent to form coated particles, wherein the surface-altering agentcomprises a triblock copolymer comprising a hydrophilicblock-hydrophobic block-hydrophilic block configuration, wherein thehydrophobic block has a molecular weight of at least about 2 kDa, andthe hydrophilic blocks constitute at least about 15 wt % of the triblockcopolymer, wherein the hydrophobic block associates with the surfaces ofthe core particles, and wherein the hydrophilic block is present at thesurfaces of the coated particles and renders the coated particleshydrophilic. The coated particles have a relative velocity of greaterthan 0.5 in mucus.

In another set of embodiments, a method of treatment is provided. Themethod comprises administering to a patient or a subject in needthereof, a composition comprising a plurality of coated particles. Thecoated particle comprises a core particle comprising a solidpharmaceutical agent or a salt thereof, wherein the agent or salt has anaqueous solubility of less than or equal to about 1 mg/mL at 25° C. atany point throughout the pH range, wherein the pharmaceutical agent orsalt thereof constitutes at least about 80 wt % of the core particle.The coated particle also includes a coating comprising asurface-altering agent surrounding the core particle, wherein thesurface-altering agent comprises a triblock copolymer comprising ahydrophilic block—hydrophobic block—hydrophilic block configuration,wherein the hydrophobic block has a molecular weight of at least about 2kDa, and the hydrophilic blocks constitute at least about 15 wt % of thetriblock copolymer, wherein the hydrophobic block associates with thesurface of the core particle, wherein the hydrophilic block is presentat the surface of the coated particle and renders the coated particlehydrophilic, and wherein the surface-altering agent is present on thesurface of the core particle at a density of at least about 0.001molecules per nanometer squared. The coated particles have a relativevelocity of greater than 0.5 in mucus.

In another set of embodiments, a method is provided. The methodcomprises delivering to a mucus membrane a composition comprising aplurality of coated particles. The coated particle comprises a coreparticle comprising a solid pharmaceutical agent or a salt thereof,wherein the agent or salt has an aqueous solubility of less than orequal to about 1 mg/mL at 25° C. at any point throughout the pH range.The pharmaceutical agent or salt thereof constitutes at least about 80wt % of the core particle. The coated particle also includes a coatingcomprising a surface-altering agent surrounding the core particle,wherein the surface-altering agent comprises a triblock copolymercomprising a hydrophilic block—hydrophobic block—hydrophilic blockconfiguration, wherein the hydrophobic block has a molecular weight ofat least about 2 kDa, and the hydrophilic blocks constitute at leastabout 15 wt % of the triblock copolymer. The hydrophobic blockassociates with the surface of the core particle, and the hydrophilicblock is present at the surface of the coated particle and renders thecoated particle hydrophilic. The surface-altering agent is present onthe surface of the core particle at a density of at least about 0.001molecules per nanometer squared. The coated particles have a relativevelocity of greater than 0.5 in mucus.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a schematic drawing of a mucus-penetrating particle having acoating and a core of a solid pharmaceutical agent according to one setof embodiments;

FIG. 2A is a plot showing the ensemble averaged velocity <V_(mean)> inhuman cervicovaginal mucus (CVM) for 200 nm carboxylated polystyreneparticles (negative control), 200 nm PEGylated polystyrene particles(positive control), and nanocrystal particles (sample) made bynanomilling and coated with different stabilizers/surface-alteringagents according to one set of embodiments;

FIG. 2B is a plot showing the relative velocity <V_(mean)>_(rel) in CVMfor nanocrystal particles made by nanomilling and coated with differentstabilizers/surface-altering agents according to one set of embodiments;

FIGS. 3A-3D are histograms showing distribution of trajectory-meanvelocity V_(mean) in CVM within an ensemble of nanocrystal particlescoated with different surface-altering agents according to one set ofembodiments;

FIG. 4 is a plot showing <V_(mean)>_(rel) in CVM for nanocrystalparticles coated with different PEO-PPO-PEO Pluronic® triblockcopolymers, mapped with respect to molecular weight of the PPO block andthe PEO weight content (%), according to one set of embodiments;

FIG. 5 is a plot showing the mass transport through CVM for solidparticles having different core materials that are coated with eitherPluronic® F127 (MPP) or sodium dodecyl sulfate (CP, a negative control),according to one set of embodiments;

FIGS. 6A-6C show drug levels of loteprednol etabonate in the palpebralconjunctiva (FIG. 6A), bulbar conjunctiva (FIG. 6B), and cornea (FIG.6C) of New Zealand white rabbits after administration of prescriptionloteprednol etabonate, Lotemax®, or particles of loteprednol etabonatethat were coated with Pluronic® F127, according to one set ofembodiments;

FIGS. 7A and 7B are physicochemical characterizations of CUR-1% F127particles according to one set of embodiments. FIG. 7A is a powder X-raydiffraction (Powder-XRD) diagram of F127, raw curcumin and CUR-1% F127particles. FIG. 7B is a representative transmission electron microscope(TEM) image of CUR-1% F127 particles.

FIGS. 8A and 8B are ensemble averaged geometric mean-squareddisplacements (<MSD>) of CUR-1% F127 particles, 200 nm carboxylatedpolystyrene (PSCOOH) and 200 nm PEGylated polystyrene (PSPEG) particlesin CVM (FIG. 8A) and human cystic fibrosis sputum (CFS) (FIG. 8B) as afunction of time scale according to one set of embodiments. Datarepresent the ensemble average of five independent experiments, withn≥100 for each experiment. Error bars indicate geometric standard error.

FIG. 9 is a plot showing geometric ensemble effective diffusivity(<Deff>) at a time scale of 1 s for CUR particles formulated indifferent concentrations of F127 in human CVM according to one set ofembodiments. Data represents the ensemble average of at least 3independent experiments, with n≥100 for each experiment. Error barsindicate geometric standard error.

FIGS. 10A-10C are plots showing diffusivity of CUR particles formulatedwith different Pluronics® in human CVM according to one set ofembodiments. FIG. 10A shows distribution of <Deff> at a time scale of iswith regards to the molecular weight (MW) of poly(ethylene glycol) (PEG)segment and poly(propylene oxide) (PPO) segment of Pluronics®. Each datapoint represents a specific type of Pluronics®. PPO and PEG MW wereestimated based on the molecular weight provided by the manufacturer.FIGS. 10 B-C show <Deff> at a time scale of 1 s as a function of the MWof (B) PEG or (C) PPO segments. Inset represents the same plot withlinear scale of <Deff>, while R represents the correlation coefficient.Data represent the ensemble average of at least three independentexperiments, with n≥100 for each experiment. Error bars indicategeometric standard error.

FIG. 11 is a plot showing cumulative release of CUR-1% F127 particles inphosphate buffered saline (pH=7.4) with octanol extraction according toone set of embodiments;

FIG. 12 is a plot showing cumulative release from a dialysis bag of freeTFV in solution vs. TFV-Zn particles in suspension into normal phosphatebuffered saline according to one set of embodiments;

FIGS. 13A-13B are images showing distribution of mucuspenetrating/F127-coated TFV particles (FIG. 13A) andmuco-adhesive/PVA-coated TFV particles (FIG. 13B) on flattened vaginaltissue from human-like estrus phase mice according to one set ofembodiments. Vaginal tissues were removed within 10 minutes ofadministration.

FIGS. 14A-14D show the transport rates of CP and MPP in mouse estrusphase CVM. (FIG. 14A) Representative trajectories for particlesexhibiting effective diffusivities within one SEM of the ensembleaverage at a time scale of 1 s. (FIG. 14B) Ensemble-averaged geometricmean square displacements (<MSD>) as a function of time scale. Data forparticles on ex vivo mouse vaginal tissue (mCVM) compared to the sameparticles in ex vivo human CVM (hCVM) (S. K. Lai, Y. Y. Wang, K. Hida,R. Cone, J. Hanes, Nanoparticles reveal that human cervicovaginal mucusis riddled with pores larger than viruses. P Natl Acad Sci USA 107,598-603 (2010)) and the theoretical diffusion rate of 110 nm particlesin water (˜4 μm²/s). (FIG. 14C) Distributions of the logarithms ofindividual particle effective diffusivities (D_(eff)) at a time scale of1 s. Data represent the ensemble average of three independentexperiments, with n≥150 particles for each experiment. Diffusivityvalues to the left of the dotted line indicate particles with MSD valuesless than the particle diameter. (FIG. 14D) Percentage of particlescapable of penetrating a 100 μm-thick layer of mouse CVM over time,based on Fick's Second Law of diffusion simulation of particlesundergoing random diffusion, with diffusivities equal to theexperimentally measured diffusivities of the particles. FIG. 14E is agraphical depiction of vaginal drug delivery from gel, CP, and MPPformulations.

FIGS. 15A-15B are plots showing the transport of nanoparticles on IEmouse vaginal tissue. Ensemble-averaged geometric mean squareddisplacements (<MDS>) as a function of time scale. (FIG. 15A) Data areshown for MPP and CP on ex vivo vaginal tissue of induced estrus (IE)and naturally cycling estrus phase mice. (FIG. 15B) Biodegradable MPPson IE tissue were compared to non-degradable MPPs. Data represent theensemble average of at least 3 independent experiments, with an averagen≥150 particles and at least 130 particles for each experiment. Data arepresented as a mean with the standard error of the mean (SEM).

FIG. 16 includes images illustrating particle distribution in the mousevagina. Distribution of red fluorescent non-biodegradable andbiodegradable CPs and MPPs in transverse cryosections of estrus phaseand IE mouse vaginal tissue. Images are representative of n≥3 mice.

FIG. 17 includes images and plots showing the quantification of vaginalnanoparticle coverage. Distribution of red fluorescent non-biodegradableand biodegradable CPs and MPPs on flattened estrus phase mouse vaginaltissue. Insets are images of dark areas at higher magnification. Imagesare representative of the averages calculated for n≥3 mice. Data aremeans±SEM. *P<0.05, Student's t test.

FIG. 18 includes images showing the cervical nanoparticle coverage.Cervical distribution of red fluorescent non-biodegradable andbiodegradable CPs and MPPs on estrus phase mouse cervical tissue. Insetsare images of dark areas at higher magnification. Images arerepresentative of n≥3 mice. Data are means±SEM. *P<0.05, Student's ttest.

FIG. 19 includes images showing the effects of mucus removal onmucoadhesive nanoparticles. Distribution of red fluorescentnon-biodegradable and biodegradable CPs in transverse cryosections ofmouse vaginal tissue with either an intact mucus layer (No treatment) ormucus removed by lavage and swabbing (Mucus removed). Images arerepresentative of n≥3 mice.

FIG. 20 includes images showing the particle distribution in the IEmouse vagina. Distribution of red fluorescent non-biodegradable CP andMPP in transverse cryosections of IE mouse vaginal tissue. Images arerepresentative of n≥3 mice.

FIGS. 21A-21B are images and a plot showing the retention ofnon-biodegradable MPPs and CPs in the IE mouse cervicovaginal tract.(FIG. 21A) Overlay of particle fluorescence intensity and bright-fieldimages for CPs and MPPs in whole cervicovaginal tract tissue. (FIG. 21B)Fraction of particles remaining over time based on quantification ofparticle fluorescence. Data are means±SEM (n≥7). *P<0.05, Student's ttest.

FIG. 22 includes images showing the distribution and retention of amodel drug, FITC, in the estrus mouse vagina delivered in gel form orencapsulated in biodegradable MPPs. Fluorescent images were taken offlattened mouse vaginal tissue after 24 h. Images are representative ofn≥3 mice. Data are means±SEM. *P<0.05, Student's t test.

FIG. 23 includes images showing the acute toxicity and cytokineconcentrations with daily administration. Hematoxylin and eosin(H&E)-stained cross-sections of mouse DP vaginal tissue excised 24 hafter intravaginal administration of 5% N9, PBS, CPs, MPPs, BD-CPs, andBD-MPPs. Scale bar applies to all images. Arrowheads point to clustersof neutrophils. Images are representative of n≥5 mice.

FIG. 24 is a plot showing the cytokine concentrations with dailyadministration. Cytokine concentrations in DP mouse cervicovaginallavage (CVL) after daily vaginal treatments for 7 days. Data aremeans±SEM. *P<0.05, Student's t test.

DETAILED DESCRIPTION

Nanocrystals, compositions, and methods that aid particle transport inmucus are provided. In some embodiments, the compositions and methodsinvolve making mucus-penetrating particles (MPP) without any polymericcarriers, or with minimal use of polymeric carriers. The compositionsand methods may include, in some embodiments, modifying the surfacecoatings of particles formed of pharmaceutical agents that have a lowwater/aqueous solubility. Such methods and compositions can be used toachieve efficient transport of particles of pharmaceutical agents thoughmucus barriers in the body for a wide spectrum of applications,including drug delivery, imaging, and diagnostic applications. Incertain embodiments, a pharmaceutical composition including suchparticles is well-suited for administration routes involving theparticles passing through a mucosal barrier.

In some embodiments, the particles described herein have a core-shelltype arrangement. The core may comprise a solid pharmaceutical agent ora salt thereof having a relatively low aqueous solubility. The core maybe coated with a coating or shell comprising a surface-altering agentthat facilitates mobility of the particle in mucus. As described in moredetail below, in some embodiments the surface-altering agent maycomprise a triblock copolymer comprising a hydrophilic block-hydrophobicblock-hydrophilic block configuration. The molecular weights of each ofthe hydrophilic and hydrophobic blocks may be chosen to impart certaintransport characteristics to the particles, such as increased transportthrough mucus.

Non-limiting examples of particles are now provided. As shown in theillustrative embodiment of FIG. 1, a particle 10 includes a core 16(which may be in the form of a particle, referred to herein as a coreparticle) and a coating 20 surrounding the core. In one set ofembodiments, a substantial portion of the core is formed of one or moresolid pharmaceutical agents (e.g., a drug, therapeutic agent, diagnosticagent, imaging agent) that can lead to certain beneficial and/ortherapeutic effects. The core may be, for example, a nanocrystal (i.e.,a nanocrystal particle) of a pharmaceutical agent. The core includes asurface 24 to which one or more surface-altering agents can be attached.For instance, in some cases, core 16 is surrounded by coating 20, whichincludes an inner surface 28 and an outer surface 32. The coating may beformed, at least in part, of one or more surface-altering agents 34,such as a polymer (e.g., a block copolymer), which may associate withsurface 24 of the core. Surface-altering agent 34 may be associated withthe core particle by, for example, being covalently attached to the coreparticle, non-covalently attached to the core particle, adsorbed to thecore, or attached to the core through ionic interactions, hydrophobicand/or hydrophilic interactions, electrostatic interactions, van derWaals interactions, or combinations thereof. In one set of embodiments,the surface-altering agents, or portions thereof, are chosen tofacilitate transport of the particle through a mucosal barrier (e.g.,mucus or a mucosal membrane).

In certain embodiments described herein, one or more surface-alteringagents 34 are oriented in a particular configuration in the coating ofthe particle. For example, in some embodiments in which asurface-altering agent is a triblock copolymer, such as a triblockcopolymer having a hydrophilic block—hydrophobic block—hydrophilic blockconfiguration, a hydrophobic block 36 may be oriented towards thesurface of the core, and hydrophilic blocks 38 may be oriented away fromthe core surface (e.g., towards the exterior of the particle). Thehydrophilic blocks may have characteristics that facilitate transport ofthe particle through a mucosal barrier, as described in more detailbelow.

Particle 10 may optionally include one or more components 40 such astargeting moieties, proteins, nucleic acids, and bioactive agents whichmay optionally impart specificity to the particle. For example, atargeting agent or molecule (e.g., a protein, nucleic acid, nucleic acidanalog, carbohydrate, or small molecule), if present, may aid indirecting the particle to a specific location in the subject's body. Thelocation may be, for example, a tissue, a particular cell type, or asubcellular compartment. One or more components 40, if present, may beassociated with the core, the coating, or both; e.g., they may beassociated with surface 24 of the core, inner surface 28 of the coating,outer surface 32 of the coating, and/or embedded in the coating. The oneor more components 40 may be associated through covalent bonds,absorption, or attached through ionic interactions, hydrophobic and/orhydrophilic interactions, electrostatic interactions, van der Waalsinteractions, or combinations thereof. In some embodiments, a componentmay be attached (e.g., covalently) to one or more of thesurface-altering agents of the coated particle using methods known tothose of ordinary skill in the art.

It should be understood that components and configurations other thanthose shown in FIG. 1 or described herein may be suitable for certainparticles and compositions, and that not all of the components shown inFIG. 1 are necessarily present in some embodiments.

In one set of embodiments, particle 10, when introduced into a subject,may interact with one or more components in the subject such as mucus,cells, tissues, organs, particles, fluids (e.g., blood), portionsthereof, and combinations thereof. In some such embodiments, the coatingof particle 10 can be designed to include surface-altering agents orother components with properties that allow favorable interactions(e.g., transport, binding, adsorption) with one or more materials fromthe subject. For example, the coating may include surface-alteringagents or other components having a certain hydrophilicity,hydrophobicity, surface charge, functional group, specificity forbinding, and/or density to facilitate or reduce particular interactionsin the subject. One specific example includes choosing a certainhydrophilicity, hydrophobicity, surface charge, functional group,specificity for binding, and/or density of one or more surface-alteringagents to reduce the physical and/or chemical interactions between theparticle and mucus of the subject, so as to enhance the mobility of theparticle through mucus. Other examples are described in more detailbelow.

In some embodiments, once a particle is successfully transported acrossa mucosal barrier (e.g., mucus or a mucosal membrane) in a subject,further interactions between the particle in the subject may take place.Interactions may take place, in some instances, through the coatingand/or the core, and may involve, for example, the exchange of materials(e.g., pharmaceutical agents, therapeutic agents, proteins, peptides,polypeptides, nucleic acids, nutrients, e.g.) from the one or morecomponents of the subject to particle 10, and/or from particle 10 to theone or more components of the subject. For example, in some embodimentsin which the core is formed of or comprises a pharmaceutical agent, thebreakdown, release and/or transport of the pharmaceutical agent from theparticle can lead to certain beneficial and/or therapeutic effects inthe subject. As such, the particles described herein can be used for thediagnosis, prevention, treatment or management of certain diseases orbodily conditions.

Specific examples for the use of the particles described herein areprovided below in the context of being suitable for administration to amucosal barrier (e.g., mucus or a mucosal membrane) in a subject. Itshould be appreciated that while many of the embodiments herein aredescribed in this context, and in the context of providing a benefit fordiseases and conditions that involve transport of materials across amucosal barrier, the invention is not limited as such and the particles,compositions, kits, and methods described herein may be used to prevent,treat, or manage other diseases or bodily conditions.

Mucus is a sticky viscoelastic gel that protects against pathogens,toxins, and debris at various points of entry into the body, includingthe eyes, nose, lungs, gastrointestinal tract, and female reproductivetract. Many synthetic nanoparticles are strongly mucoadhesive and becomeeffectively trapped in the rapidly-cleared peripheral mucus layer,vastly limiting their distribution throughout the mucosal membrane aswell as penetration toward the underlying tissue. The residence time ofthese trapped particles is limited by the turnover rate of theperipheral mucus layer, which, depending on the organ, ranges fromseconds to several hours. To ensure effective delivery of particlesincluding pharmaceutical agents (e.g., therapeutic, diagnostic, and/orimaging agents) via mucus membranes, such particles must be able toreadily diffuse through the mucus barrier, avoiding mucus adhesion.

It has been recently demonstrated that modifying surfaces of polymericnanoparticles with a mucus-penetrating coating can minimize adhesion tomucus and thus allow rapid particle penetration across mucus barriers.Specifically, it has been shown that polymeric nanoparticles as large as500 nm, when coated covalently with dense coatings of low molecularweight PEG (2 kDa −5 kDa) or non-covalently with specific Pluronic®molecules (e.g., P103, P105, F127) can penetrate human mucus nearly asfast as they move in pure water, and at rates almost 100-fold fasterthan similarly-sized uncoated polymeric particles.

Nevertheless, polymer-based mucus-penetrating particles may have one ormore inherent limitations in some embodiments. In particular, in lightof drug delivery applications, these limitations may include one or moreof the following: A) Low drug encapsulation efficiency and low drugloading: Encapsulation of drugs into polymeric particles is ofteninefficient, as generally less than 10% of the total amount of drug usedgets encapsulated into particles during manufacturing. Additionally,drug loadings above 50% are rarely achieved. B) Convenience of usage:Formulations based on drug-loaded polymeric particles, in general,typically need to be stored as dry powder to avoid premature drugrelease and, thus, require either point-of-use re-constitution or asophisticated dosing device. C) Biocompatibility: Accumulation of slowlydegrading polymer carriers following repeated dosing and their toxicityover the long term present a major concern for polymeric drug carriers.D) Chemical and physical stability: Polymer degradation may compromisestability of encapsulated drugs. In many encapsulation processes, thedrug undergoes a transition from a solution phase to a solid phase,which is not well-controlled in terms of physical form of the emergingsolid phase (i.e., amorphous vs. crystalline vs. crystallinepolymorphs). This is a concern for multiple aspects of formulationperformance, including physical and chemical stability and releasekinetics. E) Manufacturing complexity: Manufacturing, especiallyscalability, of drug-loaded polymeric MPP is a fairly complex processthat typically involves multiple steps and a considerable amount oftoxic organic solvents.

In some embodiments described herein, the compositions and methods ofmaking particles, including certain compositions and methods for makingparticles that have increased transport through mucosal barriers,address one or more, or all, of the concerns described above.Specifically, in some embodiments, the compositions and methods do notinvolve encapsulation into polymeric carriers or involve minimal use ofpolymeric carriers. Advantageously, by avoiding or minimizing the needto encapsulate pharmaceutical agents (e.g., drugs, imaging or diagnosticagents) into polymeric carriers, certain limitations of polymeric MPPwith respect to drug loading, convenience of usage, biocompatibility,stability, and/or complexity of manufacturing, may be addressed. Themethods and compositions described herein may facilitate clinicaldevelopment of the mucus-penetrating particle technology.

Core Particles

As described above in reference to FIG. 1, particle 10 may include acore 16. The core may be formed of any suitable material, such as anorganic material, an inorganic material, a polymer, or combinationsthereof. In one set of embodiments, the core comprises a solid. Thesolid may be, for example, a crystalline or an amorphous solid, such asa crystalline or amorphous solid pharmaceutical agent (e.g., atherapeutic agent, diagnostic agent, and/or imaging agent), or a saltthereof. In some embodiments, more than one pharmaceutical agents may bepresent in the core. Specific examples of pharmaceutical agents areprovided in more detail below.

The pharmaceutical agent may be present in the core in any suitableamount, e.g., at least about 1 wt %, at least about 5 wt %, at leastabout 10 wt %, at least about 20 wt %, at least about 30 wt %, at leastabout 40 wt %, at least about 50 wt %, at least about 60 wt %, at leastabout 70 wt %, at least about 80 wt %, at least about 85 wt %, at leastabout 90 wt %, at least about 95 wt %, or at least about 99 wt % of thecore. In one embodiment, the core is formed of 100 wt % of thepharmaceutical agent. In some cases, the pharmaceutical agent may bepresent in the core at less than about 100 wt %, less than about 90 wt%, less than about 80 wt %, less than about 70 wt %, less than about 60wt %, less than about 50 wt %, less than about 40 wt %, less than about30 wt %, less than about 20 wt %, less than about 10 wt %, less thanabout 5 wt %, less than about 2 wt %, or less than about 1 wt %.Combinations of the above-referenced ranges are also possible (e.g.,present in an amount of at least about 80 wt % and less than about 100wt %). Other ranges are also possible.

In embodiments in which the core particles comprise relatively highamounts of a pharmaceutical agent (e.g., at least about 50 wt % of thecore particle), the core particles generally have an increased loadingof the pharmaceutical agent compared to particles that are formed byencapsulating agents into polymeric carriers. This is an advantage fordrug delivery applications, since higher drug loadings mean that fewernumbers of particles may be needed to achieve a desired effect comparedto the use of particles containing polymeric carriers.

In some embodiments, the core is formed of a solid material having arelatively low aqueous solubility (i.e., a solubility in water,optionally with one or more buffers), and/or a relatively low solubilityin the solution in which the solid material is being coated with asurface-altering agent. For example, the solid material may have anaqueous solubility (or a solubility in a coating solution) of less thanabout or equal to about 5 mg/mL, less than or equal to about 2 mg/mL,less than or equal to about 1 mg/mL, less than or equal to about 0.5mg/mL, less than or equal to about 0.1 mg/mL, less than or equal toabout 0.05 mg/mL, less than or equal to about 0.01 mg/mL, less than orequal to about 1 μg/mL, less than or equal to about 0.1 μg/mL, less thanor equal to about 0.01 μg/mL, less than or equal to about 1 ng/mL, lessthan or equal to about 0.1 ng/mL, or less than or equal to about 0.01ng/mL at 25° C. In some embodiments, the solid material may have anaqueous solubility (or a solubility in a coating solution) of at leastabout 1 pg/mL, at least about 10 pg/mL, at least about 0.1 ng/mL, atleast about 1 ng/mL, at least about 10 ng/mL, at least about 0.1 μg/mL,at least about 1 μg/mL, at least about 5 μg/mL, at least about 0.01mg/mL, at least about 0.05 mg/mL, at least about 0.1 mg/mL, at leastabout 0.5 mg/mL, at least about 1.0 mg/mL, at least about 2 mg/mL.Combinations of the above-noted ranges are possible (e.g., an aqueoussolubility or a solubility in a coating solution of at least about 10pg/mL and less than about or equal to about 1 mg/mL). Other ranges arealso possible. The solid material may have these or other ranges ofaqueous solubilities at any point throughout the pH range (e.g., from pH1 to pH 14).

In some embodiments, the core may be formed of a material within one ofthe ranges of solubilities classified by the U.S. PharmacopeiaConvention: e.g., very soluble: >1,000 mg/mL; freely soluble: 100-1,000mg/mL; soluble: 33-100 mg/mL; sparingly soluble: 10-33 mg/mL; slightlysoluble: 1-10 mg/mL; very slightly soluble: 0.1-1 mg/mL; and practicallyinsoluble: <0.1 mg/mL.

Although a core may be hydrophobic or hydrophilic, in many embodimentsdescribed herein, the core is substantially hydrophobic. “Hydrophobic”and “hydrophilic” are given their ordinary meaning in the art and, aswill be understood by those skilled in the art, in many instancesherein, are relative terms. Relative hydrophobicities andhydrophilicities of materials can be determined by measuring the contactangle of a water droplet on a planar surface of the substance to bemeasured, e.g., using an instrument such as a contact angle goniometerand a packed powder of the core material.

In some embodiments, a material (e.g., a material forming a particlecore) has a contact angle of at least about 20 degrees, at least about30 degrees, at least about 40 degrees, at least about 50 degrees, atleast about 60 degrees, at least about 70 degrees, at least about 80degrees, at least about 90 degrees, at least about 100 degrees, at leastabout 110 degrees, at least about 120 degrees, or at least about 130degrees. In some embodiments, a material has a contact angle of lessthan or equal to about 160 degrees, less than or equal to about 150degrees, less than or equal to about 140 degrees, less than or equal toabout 130 degrees, less than or equal to about 120 degrees, less than orequal to about 110 degrees, less than or equal to about 100 degrees,less than or equal to about 90 degrees, less than or equal to about 80degrees, or less than or equal to about 70 degrees. Combinations of theabove-referenced ranges are also possible (e.g., a contact angle of atleast about 30 degrees and less than or equal to about 120 degrees).Other ranges are also possible.

Contact angle measurements can be made using a variety of techniques;here a static contact angle measurement between a pellet of the startingmaterial which will be used to form the core and a bead of water isreferenced. The material used to form the core was received as a finepowder or otherwise was ground into a fine powder using a mortar andpestle. In order to form a surface on which to make measurements, thepowder was packed using a 7 mm pellet die set from International CrystalLabs. The material was added to the die and pressure was applied by handto pack the powder into a pellet, no pellet press or high pressure wasused. The pellet was then suspended for testing so that the top andbottom of the pellet (defined as the surface water is added to and theopposite parallel surface respectively) were not in contact with anysurface. This was done by not fully removing the pellet from the collarof the die set. The pellet therefore touches the collar on the sides andmakes no contact on the top or bottom. For contact angle measurements,water was added to the surface of the pellet until a bead of water witha steady contact angle over 30 seconds was obtained. The water was addedinto the bead of water by submerging or contacting the tip of thepipette or syringe used for addition to the bead of water. Once a stablebead of water was obtained, an image was taken and the contact angle wasmeasured using standard practices.

In embodiments in which the core comprises an inorganic material (e.g.,for use as imaging agents), the inorganic material may include, forexample, a metal (e.g., Ag, Au, Pt, Fe, Cr, Co, Ni, Cu, Zn, and othertransition metals), a semiconductor (e.g., silicon, silicon compoundsand alloys, cadmium selenide, cadmium sulfide, indium arsenide, andindium phosphide), or an insulator (e.g., ceramics such as siliconoxide). The inorganic material may be present in the core in anysuitable amount, e.g., at least about 1 wt %, at least about 5 wt %, atleast about 10 wt %, at least about 20 wt %, at least about 30 wt %, atleast about 40 wt %, at least about 50 wt %, at least about 75 wt %, atleast about 90 wt %, or at least about 99 wt %. In one embodiment, thecore is formed of 100 wt % inorganic material. In some cases, theinorganic material may be present in the core at less than about 100 wt%, less than about 90 wt %, less than about 80 wt %, less than about 70wt %, less than about 60 wt %, less than about 50 wt %, less than about40 wt %, less than about 30 wt %, less than about 20 wt %, less thanabout 10 wt %, less than about 5 wt %, less than about 2 wt %, or lessthan about 1 wt %. Combinations of the above-referenced ranges are alsopossible (e.g., present in an amount of at least about 1 wt % and lessthan about 20 wt %). Other ranges are also possible.

The core may, in some cases, be in the form of a quantum dot, a carbonnanotube, a carbon nanowire, or a carbon nanorod. In some cases, thecore comprises, or is formed of, a material that is not of biologicalorigin.

In some embodiments, the core includes one or more organic materialssuch as a synthetic polymer and/or a natural polymer. Examples ofsynthetic polymers include non-degradable polymers such aspolymethacrylate and degradable polymers such as polylactic acid,polyglycolic acid and copolymers thereof. Examples of natural polymersinclude hyaluronic acid, chitosan, and collagen. Other examples ofpolymers that may be suitable for portions of the core include thoseherein suitable for forming coatings on particles, as described below. Apolymer may be present in the core in any suitable amount, e.g., lessthan about 100 wt %, less than about 90 wt %, less than about 80 wt %,less than about 70 wt %, less than about 60 wt %, less than about 50 wt%, less than about 40 wt %, less than about 30 wt %, less than about 20wt %, less than about 10 wt %, less than about 5 wt %, less than about 2wt %, or less than about 1 wt %. In some cases, the polymer may bepresent in an amount of at least about 1 wt %, at least about 5 wt %, atleast about 10 wt %, at least about 20 wt %, at least about 30 wt %, atleast about 40 wt %, at least about 50 wt %, at least about 75 wt %, atleast about 90 wt %, or at least about 99 wt % in the core. Combinationsof the above-referenced ranges are also possible (e.g., present in anamount of at least about 1 wt % and less than about 20 wt %). Otherranges are also possible. In one set of embodiments, the core is formedis substantially free of a polymeric component.

The core may have any suitable shape and/or size. For instance, the coremay be substantially spherical, non-spherical, oval, rod-shaped,pyramidal, cube-like, disk-shaped, wire-like, or irregularly shaped. Thecore may have a largest or smallest cross-sectional dimension of, forexample, less than or equal to about 10 μm, less than or equal to about5 μm, less than or equal to about 1 μm, less than or equal to about 800nm, less than or equal to about 700 nm, less than or equal to about 500nm, less than or equal to 400 nm, less than or equal to 300 nm, lessthan or equal to about 200 nm, less than or equal to about 100 nm, lessthan or equal to about 75 nm, less than or equal to about 50 nm, lessthan or equal to about 40 nm, less than or equal to about 35 nm, lessthan or equal to about 30 nm, less than or equal to about 25 nm, lessthan or equal to about 20 nm, less than or equal to about 15 nm, or lessthan or equal to about 5 nm. In some cases, the core may have a largestor smallest cross-sectional dimension of, for example, at least about 5nm, at least about 20 nm, at least about 50 nm, at least about 100 nm,at least about 200 nm, at least about 300 nm, at least about 400 nm, atleast about 500 nm, at least about 1 μm, or at least about 5 μm.Combinations of the above-referenced ranges are also possible (e.g., alargest or smallest cross-sectional dimension of at least about 50 nmand less than about 500 nm). Other ranges are also possible. In someembodiments, the sizes of the cores formed by a process described hereinhave a Gaussian-type distribution. Unless indicated otherwise, themeasurements of particle/core sizes herein refer to the smallestcross-sectional dimension.

Those of ordinary skill in the art are familiar with techniques todetermine sizes (e.g., smallest or largest cross-sectional dimensions)of particles. Examples of suitable techniques include (DLS),transmission electron microscopy, scanning electron microscopy,electroresistance counting and laser diffraction. Other suitabletechniques are known to those or ordinary skill in the art. Althoughmany methods for determining sizes of particles are known, the sizesdescribed herein (e.g., average particle sizes, thicknesses) refer toones measured by dynamic light scattering.

Methods of Forming Core Particles and Coated Particles

The core particles described herein may be formed by any suitablemethod. In some embodiments, a milling process is used to reduce thesize of a solid material to form particles in the micrometer tonanometer size range. Dry and wet milling processes such as jet milling,cryo-milling, ball milling, media milling, and homogenization are knownand can be used in methods described herein. Generally, in a wet millingprocess, a suspension of the material to be used as the core is mixedwith milling media with or without excipients to reduce particle size.Dry milling is a process wherein the material to be used as the core ismixed with milling media with or without excipients to reduce particlesize. In a cryo-milling process, a suspension of the material to be usedas the core is mixed with milling media with or without excipients undercooled temperatures.

In some embodiments, the core particles described herein may be producedby nanomilling of a solid material (e.g., a pharmaceutical agent) in thepresence of one or more stabilizers/surface-altering agents. Smallparticles of a solid material may require the presence of one or morestabilizers/surface-altering agents, particularly on the surface of theparticles, in order to stabilize a suspension of particles withoutagglomeration or aggregation in a liquid solution. In some suchembodiments, the stabilizer may act as a surface-altering agent, forminga coating on the particle.

As described herein, in some embodiments, a method of forming a coreparticle involves choosing a stabilizer that is suitable for bothnanomilling and for forming a coating on the particle and rendering theparticle mucus penetrating. For example, as described in more detailbelow, it has been demonstrated that 200-500 nm nanoparticles of a modelcompound pyrene produced by nanomilling of pyrene in the presence ofPluronic® F127 resulted in particles that can penetrate physiologicalmucus samples at the same rate as well-established polymer-based MPP.Interestingly, it was observed that only a handful ofstabilizers/surface-altering agents tested fit the criteria of beingsuitable for both nanomilling and for forming a coating on the particlethat renders the particle mucus penetrating, as described in more detailbelow.

In a wet milling process, milling can be performed in a dispersion(e.g., an aqueous dispersion) containing one or more stabilizers (e.g.,a surface-altering agent), a grinding medium, a solid to be milled(e.g., a solid pharmaceutical agent), and a solvent. Any suitable amountof a stabilizer/surface-altering agent can be included in the solvent.In some embodiments, a stabilizer/surface-altering agent may be presentin the solvent in an amount of at least about 0.001% (wt % or % weightto volume (w:v)), at least about 0.01%, at least about 0.1%, at leastabout 0.5%, at least about 1%, at least about 2%, at least about 3%, atleast about 4%, at least about 5%, at least about 6%, at least about 7%,at least about 8%, at least about 10%, at least about 12%, at leastabout 15%, at least about 20%, at least about 40%, at least about 60%,or at least about 80% of the solvent. In some cases, the stabilizer maybe present in the solvent in an amount of about 100% (e.g., in aninstance where the stabilizer/surface-altering agent is the solvent). Inother embodiments, the stabilizer may be present in the solvent in anamount of less than or equal to about 100%, less than or equal to about80%, less than or equal to about 60%, less than or equal to about 40%,less than or equal to about 20%, less than or equal to about 15%, lessthan or equal to about 12%, less than or equal to about 10%, less thanor equal to about 8%, less than or equal to about 7%, less than or equalto about 6%, less than or equal to about 5%, less than or equal to about4%, less than or equal to about 3%, less than or equal to about 2%, orless than or equal to about 1% of the solvent. Combinations of theabove-referenced ranges are also possible (e.g., an amount of less thanor equal to about 5% and at least about 1% of the solvent). Other rangesare also possible. The particular range chosen may influence factorsthat may affect the ability of the particles to penetrate mucus such asthe stability of the coating of the stabilizer/surface-altering agent onthe particle surface, the average thickness of the coating of thestabilizer/surface-altering agent on the particles, the orientation ofthe stabilizer/surface-altering agent on the particles, the density ofthe stabilizer/surface altering agent on the particles, stabilizer:drugratio, drug concentration, the size and polydispersity of the particlesformed, and the morphology of the particles formed.

The pharmaceutical agent (or salt thereof) may be present in the solventin any suitable amount. In some embodiments, the pharmaceutical agent(or salt thereof) is present in an amount of at least about 0.001% (wt %or % weight to volume (w:v)), at least about 0.01%, at least about 0.1%,at least about 0.5%, at least about 1%, at least about 2%, at leastabout 3%, at least about 4%, at least about 5%, at least about 6%, atleast about 7%, at least about 8%, at least about 10%, at least about12%, at least about 15%, at least about 20%, at least about 40%, atleast about 60%, or at least about 80% of the solvent. In some cases,the pharmaceutical agent (or salt thereof) may be present in the solventin an amount of less than or equal to about 100%, less than or equal toabout 90%, less than or equal to about 80%, less than or equal to about60%, less than or equal to about 40%, less than or equal to about 20%,less than or equal to about 15%, less than or equal to about 12%, lessthan or equal to about 10%, less than or equal to about 8%, less than orequal to about 7%, less than or equal to about 6%, less than or equal toabout 5%, less than or equal to about 4%, less than or equal to about3%, less than or equal to about 2%, or less than or equal to about 1% ofthe solvent. Combinations of the above-referenced ranges are alsopossible (e.g., an amount of less than or equal to about 20% and atleast about 1% of the solvent). In some embodiments, the pharmaceuticalagent is present in the above ranges but in w:v

The ratio of stabilizer/surface-altering agent to pharmaceutical agent(or salt thereof) in a solvent may also vary. In some embodiments, theratio of stabilizer/surface-altering agent to pharmaceutical agent (orsalt thereof) may be at least 0.001:1 (weight ratio, molar ratio, or w:vratio), at least 0.01:1, at least 0.01:1, at least 1:1, at least 2:1, atleast 3:1, at least 5:1, at least 10:1, at least 25:1, at least 50:1, atleast 100:1, or at least 500:1. In some cases, the ratio ofstabilizer/surface-altering agent to pharmaceutical agent (or saltthereof) may be less than or equal to 1000:1 (weight ratio or molarratio), less than or equal to 500:1, less than or equal to 100:1, lessthan or equal to 75:1, less than or equal to 50:1, less than or equal to25:1, less than or equal to 10:1, less than or equal to 5:1, less thanor equal to 3:1, less than or equal to 2:1, less than or equal to 1:1,or less than or equal to 0.1:1. Combinations of the above-referencedranges are possible (e.g., a ratio of at least 5:1 and less than orequal to 50:1). Other ranges are also possible.

Stabilizers/surface-altering agents may be, for example, polymers orsurfactants. Examples of polymers are those suitable for use incoatings, as described in more detail below. Non-limiting examples ofsurfactants include L-α-phosphatidylcholine (PC),1,2-dipalmitoylphosphatidycholine (DPPC), oleic acid, sorbitantrioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylenesorbitan monolaurate, polyoxyethylene sorbitan monooleate, naturallecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether,lauryl polyoxyethylene ether, block copolymers of oxyethylene andoxypropylene, synthetic lecithin, diethylene glycol dioleate,tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glycerylmonooleate, glyceryl monostearate, glyceryl monoricinoleate, cetylalcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridiniumchloride, benzalkonium chloride, olive oil, glyceryl monolaurate, cornoil, cotton seed oil, and sunflower seed oil.

It should be appreciated that while in some embodiments the stabilizerused for milling forms a coating on a particle surface, which coatingrenders particle mucus penetrating, in other embodiments, the stabilizermay be exchanged with one or more other surface-altering agents afterthe particle has been formed. For example, in one set of methods, afirst stabilizer/surface-altering agent may be used during a millingprocess and may coat a surface of a core particle, and then all orportions of the first stabilizer/surface-altering agent may be exchangedwith a second stabilizer/surface-altering agent to coat all or portionsof the core particle surface. In some cases, the secondstabilizer/surface-altering agent may render the particle mucuspenetrating more than the first stabilizer/surface-altering agent. Insome embodiments, a core particle having a coating including multiplesurface-altering agents may be formed.

Any suitable grinding medium can be used for milling. In someembodiments, a ceramic and/or polymeric material and/or a metal can beused. Examples of suitable materials may include zirconium oxide,silicon carbide, silicon oxide, silicon nitride, zirconium silicate,yttrium oxide, glass, alumina, alpha-alumina, aluminum oxide,polystyrene, poly(methyl methacrylate), titanium, steel. A grindingmedium may have any suitable size. For example, the grinding medium mayhave an average diameter of at least about 0.1 mm, at least about 0.2mm, at least about 0.5 mm, at least about 0.8 mm, at least about 1 mm,at least about 2 mm, or at least about 5 mm. In some cases, the grindingmedium may have an average diameter of less than or equal to about 5 mm,less than or equal to about 2 mm, less than or equal to about 1 mm, lessthan or equal to about 0.8, less than or equal to about 0.5 mm, or lessthan or equal to about 0.2 mm. Combinations of the above-referencedranges are also possible (e.g., an average diameter of at least about0.5 millimeters and less than or equal to about 1 mm). Other ranges arealso possible.

Any suitable solvent may be used for milling. The choice of solvent maydepend on factors such as the solid material (e.g., pharmaceuticalagent) being milled, the particular type of stabilizer/surface-alteringagent being used (e.g., one that may render the particle mucuspenetrating), the grinding material be used, among other factors.Suitable solvents may be ones that do not substantially dissolve thesolid material or the grinding material, but dissolve thestabilizer/surface-altering agent to a suitable degree. Non-limitingexamples of solvents may include water, buffered solutions, otheraqueous solutions, alcohols (e.g., ethanol, methanol, butanol), andmixtures thereof that may optionally include other components such aspharmaceutical excipients, polymers, pharmaceutical agents, salts,preservative agents, viscosity modifiers, tonicity modifier, tastemasking agents, antioxidants, pH modifier, and other pharmaceuticalexcipients. In other embodiments, an organic solvent can be used. Apharmaceutical agent may have any suitable solubility in these or othersolvents, such as a solubility in one or more of the ranges describedabove for aqueous solubility or for solubility in a coating solution.

In other embodiments, core particles may be formed by a precipitationtechnique. Precipitation techniques (e.g., microprecipitationtechniques, nanoprecipitation techniques) may involve forming a firstsolution comprising the material to be used as the core (e.g., apharmaceutical agent) and a solvent, wherein the material issubstantially soluble in the solvent. The solution may be added to asecond solution comprising another solvent in which the material issubstantially insoluble, thereby forming a plurality of particlescomprising the material. In some cases, one or more surface-alteringagents, surfactants, materials, and/or bioactive agents may be presentin the first and/or second solutions. A coating may be formed during theprocess of precipitating the core (e.g., the precipitating and coatingsteps may be performed substantially simultaneously). In otherembodiments, the particles are first formed using a precipitationtechnique, following by coating of the particles with a surface-alteringagent.

In some embodiments, a precipitation technique may be used to formparticles (e.g., nanocrystals) of a salt of a pharmaceutical agent.Generally, a precipitation technique involves dissolving the material tobe used as the core in a solvent, which is then added to a miscibleanti-solvent with or without excipients to form the core particle. Thistechnique may be useful for preparing particles of pharmaceutical agentsthat are soluble in aqueous solutions (e.g., agents having a relativelyhigh aqueous solubility). In some embodiments, pharmaceutical agentshaving one or more charged or ionizable groups can interact with acounter ion (e.g., a cation or an anion) to form a salt complex. Forexample, the pharmaceutical agent tenofovir (TFV) interacts verystrongly with zinc cations via the phosphonate group and the purine ringstructure. This interaction with zinc causes TFV precipitation intocrystals that can be stabilized with the coatings described herein,halting aggregation.

A variety of counter ions can be used to form salt complexes, includingmetals (e.g., alkali metals, alkali earth metals and transition metals).Non-limiting examples of cationic counter ions include zinc, calcium,aluminum, zinc, barium, magnesium, and copper. Non-limiting examples ofanionic counter ions include phosphate, carbonate, and fatty acids.Counter ions may be, for example, monovalent, divalent, or trivalent.Other counter ions are known in the art and can be used in theembodiments described herein.

A variety of different acids may be used in a precipitation process. Insome embodiments, a suitable acid may include deconoic acid, hexanoicacid, mucic acid, octanoic acid. In other embodiments, a suitable acidmay include acetic acid, adipic acid, L-ascorbic acid, L-aspartic acid,capric acid (decanoic acid), carbonic acid, citric acid, fumaric acid,galactaric acid, D-glucoheptonic acid, D-gluconic acid, D-glucuronicacid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolicacid, hippuric acid, hydrochloric acid, DL-lactic acid, lauric acid,maleic acid, (−)-L-malic acid, palmitic acid, phosphoric acid, sebacicacid, stearic acid, succinic acid, sulfuric acid, (+)-L-tartaric acid,or thiocyanic acid. In other embodiments, a suitable acid may includealginic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid,caprylic acid (octanoic acid), cyclamic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid, ethanesulfonic acid,2-hydroxy-, gentisic acid, glutaric acid, 2-oxo-, isobutyric acid,lactobionic acid, malonic acid, methanesulfonic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,2-naphthoic acid, 1-hydroxy-, nicotinic acid, oleic acid, orotic acid,oxalic acid, pamoic acid, (embonic acid), propionic acid,(−)-L-pyroglutamic acid, or p-toluenesulfonic acid. In yet otherembodiments, a suitable acid may include acetic acid, 2,2-dichloro-,benzoic acid, 4-acetamido-, (+)-camphor-10-sulfonic acid, caproic acid(hexanoic acid), cinnamic acid, formic acid, hydrobromic acid,DL-mandelic acid, nitric acid, salicylic acid, salicylic acid, 4-amino-,or undecylenic acid (undec-10-enoic acid). Mixtures of one or more suchacids can also be used.

A variety of different bases may be used in a precipitation process. Insome embodiments, a suitable base includes ammonia, L-arginine, calciumhydroxide, choline, glucamine, N-methyl-, lysine, magnesium hydroxide,potassium hydroxide, or sodium hydroxide. In other embodiments, asuitable base may include benethamine, benzathine, betaine, deanol,diethylamine, ethanol, 2-(diethylamino)-, hydrabamine, morpholine,4-(2-hydroxyethyl)-, pyrrolidine, 1-(2-hyroxyethyl)-, or tromethamine.In other embodiments, a suitable base may include diethanolamine(2,2′-iminobis(ethanol)), ethanolamine (2-aminoethanol),ethylenediamine, 1H-imidazole, piperazine, triethanolamine(2,2′,2″-nitrilotris(ethanol)), or zinc hydroxide. Mixtures of one ormore such bases can also be used.

Any suitable solvent can be used for precipitation, including thesolvents described herein that may be used for milling. In one set ofembodiments, an aqueous solution is used (e.g., water, bufferedsolutions, other aqueous solutions, alcohols (e.g., ethanol, methanol,butanol), and mixtures thereof that may optionally include othercomponents such as pharmaceutical excipients, polymers, andpharmaceutical agents.

In the precipitation process, the salt may have a lower aqueoussolubility (or solubility in the solvent containing the salt) than thepharmaceutical agent in the non-salt form. The aqueous solubility (orsolubility in the solvent) of the salt may be, for example, less thanabout or equal to about 5 mg/mL, less than or equal to about 2 mg/mL,less than or equal to about 1 mg/mL, less than or equal to about 0.5mg/mL, less than or equal to about 0.1 mg/mL, less than or equal toabout 0.05 mg/mL, or less than or equal to about 0.01 mg/mL, less thanor equal to about 1 μg/mL, less than or equal to about 0.1 μg/mL, lessthan or equal to about 0.01 μg/mL, less than or equal to about 1 ng/mL,less than or equal to about 0.1 ng/mL, or less than or equal to about0.01 ng/mL at 25° C. In some embodiments, the salt may have an aqueoussolubility (or solubility in the solvent) of at least about 1 pg/mL, atleast about 10 pg/mL, at least about 0.1 ng/mL, at least about 1 ng/mL,at least about 10 ng/mL, at least about 0.1 μg/mL, at least about 1μg/mL, at least about 5 μg/mL, at least about 0.01 mg/mL, at least about0.05 mg/mL, at least about 0.1 mg/mL, at least about 0.5 mg/mL, at leastabout 1.0 mg/mL, at least about 2 mg/mL. Combinations of the above-notedranges are possible (e.g., an aqueous solubility (or solubility in thesolvent) of at least about 0.001 mg/mL and less than about or equal toabout 1 mg/mL). Other ranges are also possible. The salt may have theseor other ranges of aqueous solubilities at any point throughout the pHrange (e.g., from pH 1 to pH 14).

In some embodiments, the solvent used for precipitation includes one ormore surface-altering agents as described herein, and a coating of theone or more surface-altering agents may be formed around the particle asit precipitates out of solution. The surface-altering agent may bepresent in the solvent at any suitable concentration, such as aconcentration of at least about 0.001% (w/v), at least about 0.005%(w/v), at least about 0.01% (w/v), at least about 0.05% (w/v), at leastabout 0.1% (w/v), at least about 0.5% (w/v), at least about 1% (w/v), orat least about 5% (w/v) in the aqueous solution. In some instances, thesurface-altering agent is present in the solvent at a concentration ofless than or equal to about 5% (w/v), less than or equal to about 1%(w/v), less than or equal to about 0.5% (w/v), less than or equal toabout 0.1% (w/v), less than or equal to about 0.05% (w/v), less than orequal to about 0.01% (w/v), or less than or equal to about 0.005% (w/v).Combinations of the above-referenced ranges are also possible (e.g., aconcentration of at least about 0.01 (w/v) and less than or equal toabout 1% (w/v). Other ranges are also possible.

Another exemplary method of forming a core particle includes afreeze-drying technique. In this technique, a pharmaceutical agent orsalt thereof may be dissolved in an aqueous solution, optionallycontaining a surface-altering agent. A counter ion may be added to thesolution, and the solution may be immediately flash frozen and freezedried. Dry powder can be reconstituted in a suitable solvent (e.g., anaqueous solution such as water) at a desired concentration.

A counter ion may be added to a solvent for freeze-drying in anysuitable range. In some cases, the ratio of counter ion topharmaceutical agent (e.g., salt) may be at least 0.1:1 (weight ratio ormolar ratio), at least 1:1, at least 2:1, at least 3:1, at least 5:1, atleast 10:1, at least 25:1, at least 50:1, or at least 100:1. In somecases, the ratio of counter ion to pharmaceutical agent (e.g., salt) maybe less than or equal to 100:1 (weight ratio or molar ratio), less thanor equal to 75:1, less than or equal to 50:1, less than or equal to25:1, less than or equal to 10:1, less than or equal to 5:1, less thanor equal to 3:1, less than or equal to 2:1, less than or equal to 1:1,or less than or equal to 0.1:1. Combinations of the above-referencedranges are possible (e.g., a ratio of at least 5:1 and less than orequal to 50:1). Other ranges are also possible.

If the surface-altering agent is present in the solvent prior to freezedrying, it may be present at any suitable concentration, such as aconcentration of at least about 0.001% (w/v), at least about 0.005%(w/v), at least about 0.01% (w/v), at least about 0.05% (w/v), at leastabout 0.1% (w/v), at least about 0.5% (w/v), at least about 1% (w/v), orat least about 5% (w/v) in the aqueous solution. In some instances, thesurface-altering agent is present in the solvent at a concentration ofless than or equal to about 5% (w/v), less than or equal to about 1%(w/v), less than or equal to about 0.5% (w/v), less than or equal toabout 0.1% (w/v), less than or equal to about 0.05% (w/v), less than orequal to about 0.01% (w/v), or less than or equal to about 0.005% (w/v).Combinations of the above-referenced ranges are also possible (e.g., aconcentration of at least about 0.01% (w/v) and less than or equal toabout 1% (w/v). Other ranges are also possible.

The concentration of surface-altering agent present in the solvent maybe above or below the critical micelle concentration (CMC) of thesurface-altering agent, depending on the particular surface-alteringagent used. For example, as described in the Examples section, F127concentrations both above (1%) and below (0.08%) the CMC of F127 can beused to coat stable nanocrystal particles of the pharmaceutical agenttenofovir. However, nanocrystal particles of acyclovir monophosphatewere much more sensitive to surfactant concentration, and stablenanocrystal particles could only be formed when using F127concentrations below the CMC (˜0.1%).

In other embodiments, stable particles can be formed by adding excesscounter ion to a solution containing a pharmaceutical agent. Theprecipitate can then be washed by various methods such ascentrifugation. The resultant slurry may be sonicated. One or moresurface-altering agents may be added to stabilize the resultantparticles.

Other methods of forming particles of a pharmaceutical agent are alsopossible. So called top-down techniques include, for example, millingtechniques and high pressure homogenization. In high pressurehomogenization, a suspension of the material to be used as the core isforced under pressure through a gap, valve, or aperture in order toreduce particle size. So called bottom-up techniques include, forexample, precipitation, emulsification, wherein the material to be usedas the core dissolved in a solvent is added to an immiscibleanti-solvent with or without excipients to form the core particle; andspray drying, wherein a solution of the material to be used as the corein sprayed into a gas phase anti-solvent to form the core particle.Combinations of the methods described herein and other methods are alsopossible. For example, in some embodiments, a core of a pharmaceuticalagent is first formed by precipitation, and then the size of the core isfurther reduced by a milling process.

Following formation of particles of a pharmaceutical agent, theparticles may be optionally exposed to a solution comprising a (second)surface-altering agent that may associate with and/or coat theparticles. In embodiments in which the pharmaceutical agent alreadyincludes a coating of a first surface-altering agent, all or portions ofa second surface-altering agent may be exchanged with a secondstabilizer/surface-altering agent to coat all or portions of theparticle surface. In some cases, the second surface-altering agent mayrender the particle mucus penetrating more than the firstsurface-altering agent. In other embodiments, a particle having acoating including multiple surface-altering agents may be formed (e.g.,in a single layer or in multiple layers). In other embodiments, aparticle having multiple coatings (e.g., each coating optionallycomprising different surface-altering agents) may be formed. In somecases, the coating is in the form of a monolayer of a surface-alteringagent. Other configurations are also possible.

In any of the methods described herein, a particle may be coated with asurface-altering agent by incubating the particle in a solution with thesurface-altering agent for a period of at least about 1 minutes, atleast about 2 minutes, at least about 5 min., at least about 10 min., atleast about 15 min., at least about 20 min., at least about 30 min., atleast about 60 min., or more. In some cases, incubation may take placefor a period of less than or equal to about 10 hours, less than or equalto about 5 hours, or less than or equal to about 60 min. Combinations ofthe above referenced ranges are also possible (e.g., an incubationperiod of less than or equal to 60 min. and at least about 2 min.).

Particle Coatings

As shown in the embodiment illustrated in FIG. 1, core 16 may besurrounded by coating 20 comprising one or more surface-altering agents.The particular chemical makeup and/or components of the coating andsurface-altering agent(s) can be chosen so as to impart certainfunctionality to the particles, such as enhanced transport throughmucosal barriers.

It should be understood that a coating which surrounds a core need notcompletely surround the core, although such embodiments may be possible.For example, the coating may surround at least about 10%, at least about30%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 99% of thesurface area of a core. In some cases, the coating substantiallysurrounds a core. In other cases, the coating completely surrounds acore. In other embodiments, a coating surrounds less than or equal toabout 100%, less than or equal to about 90%, less than or equal to about80%, less than or equal to about 70%, less than or equal to about 60%,or less than or equal to about 50% of the surface area of a core.Combinations of the above-referenced ranges are also possible (e.g.,surrounding greater than 80% and less than 100% of the surface area of acore).

The components of the coating may be distributed evenly across a surfaceof the core in some cases, and unevenly in other cases. For example, thecoating may include portions (e.g., holes) that do not include anymaterial in some cases. If desired, the coating may be designed to allowpenetration and/or transport of certain molecules and components into orout of the coating, but may prevent penetration and/or transport ofother molecules and components into or out of the coating. The abilityof certain molecules to penetrate and/or be transported into and/oracross a coating may depend on, for example, the packing density of thesurface-altering agents forming the coating and the chemical andphysical properties of the components forming the coating. As describedherein, the coating may include one layer of material, or multilayers ofmaterials in some embodiments. A single type of surface-altering agentmay be present, or multiple types of surface-altering agent.

A coating of a particle can have any suitable thickness. For example, acoating may have an average thickness of at least about 1 nm, at leastabout 5 nm, at least about 10 nm, at least about 30 nm, at least about50 nm, at least about 100 nm, at least about 200 nm, at least about 500nm, at least about 1 μm, or at least about 5 μm. In some cases, theaverage thickness of a coating is less than or equal to about 5 μm, lessthan or equal to about 1 μm, less than or equal to about 500 nm, lessthan or equal to about 200 nm, less than or equal to about 100 nm, lessthan a to about 50 nm, less than or equal to about 30 nm, less than orequal to about 10 nm, or less than or equal to about 5 nm. Combinationsof the above-referenced ranges are also possible (e.g., an averagethickness of at least about 1 nm and less than or equal to about 100nm). Other ranges are also possible. For particles having multiplecoatings, each coating layer may have one of the thicknesses describedabove.

In some embodiments, the compositions and methods described herein mayallow for the coating of a core particle with hydrophilicsurface-alternating moieties without requiring covalent linking of thesurface-altering moieties to the core surface. In some such embodiments,a core having a hydrophobic surface may be coated with a polymerdescribed herein, thereby causing a plurality of surface-alteringmoieties to be on the core surface without substantially altering thecharacteristics of the core itself. In other embodiments, however, asurface-altering agent is covalently linked to a core particle.

The coating and/or surface-altering agent of a particle described hereinmay comprise any suitable material, such as a hydrophobic material, ahydrophilic material, and/or an amphiphilic material. In someembodiments, the coating includes a polymer. In certain embodiments, thepolymer is a synthetic polymer (i.e., a polymer not produced in nature).In other embodiments, the polymer is a natural polymer (e.g., a protein,polysaccharide, rubber). In certain embodiments, the polymer is asurface active polymer. In certain embodiments, the polymer is anon-ionic polymer. In certain embodiments, the polymer is a non-ionicblock copolymer. In some embodiments, the polymer may be a diblockcopolymer, a triblock copolymer, e.g., e.g., where one block is ahydrophobic polymer and another block is a hydrophilic polymer. Thepolymer may be charged or uncharged.

In some embodiments, the particles described herein include a coatingcomprising a block copolymer having a relatively hydrophilic block and arelatively hydrophobic block. In some cases, the hydrophilic blocks maybe substantially present at the outer surface of the particle. Forexample, the hydrophilic blocks may form a majority of the outer surfaceof the coating and may help stabilize the particle in an aqueoussolution containing the particle. The hydrophobic block may besubstantially present in the interior of the coating and/or at thesurface of the core particle, e.g., to facilitate attachment of thecoating to the core. In some instances, the coating comprises asurface-altering agent including a triblock copolymer, wherein thetriblock copolymer comprises a hydrophilic block-hydrophobicblock-hydrophilic block configuration.

The molecular weight of the hydrophilic blocks and the hydrophobicblocks of the triblock copolymers may be selected so as to reduce themucoadhesion of a core and to ensure sufficient association of thetriblock copolymer with the core, respectively. As described herein, themolecular weight of the hydrophobic block of the triblock copolymer maybe chosen such that adequate association of the triblock copolymer withthe core occurs, thereby increasing the likelihood that the triblockcopolymer remains adhered to the core. Surprisingly, it was discoveredthat in certain embodiments, too low of a molecular weight of thehydrophobic blocks of the triblock copolymer (e.g., less than about 2kDa) does not allow for sufficient adhesion between a hydrophobic coreand the triblock copolymer, and thus, particles with such hydrophobicblocks may not exhibit sufficient reduced mucoadhesion.

In certain embodiments, the molecular weight of a hydrophobic block of atriblock copolymer (e.g., the PPO block of the triblock copolymerPEG-PPO-PEG, where the PEG block may be interchanged with PEO block) isat least about 2 kDa, at least about 3 kDa, at least about 4 kDa, atleast about 5 kDa, at least about 6 kDa, at least about 10 kDa, at leastabout 20 kDa, or at least about 50 kDa. In some embodiments, themolecular weight of the hydrophobic block is less than or equal to about100 kDa, less than or equal to about 80 kDa, less than or equal to about50 kDa, less than or equal to about 20 kDa, less than or equal to about15 kDa, less than or equal to about 13 kDa, less than or equal to about12 kDa, less than or equal to about 10 kDa, less than or equal to about8 kDa, or less than or equal to about 6 kDa. Combinations of theabove-mentioned ranges are also possible (e.g., at least about 3 kDa andless than or equal to about 15 kDa). Other ranges are also possible.

It was also discovered that in certain embodiments, a sufficient amountof the hydrophilic blocks (as a function of the total weight of thepolymer) was needed for the particles to exhibit sufficient reducedmucoadhesion. For example, in certain embodiments, hydrophilic blocksmaking up at least about 15 wt % (e.g., at least about 20 wt %, at leastabout 25 wt %, or at least about 30 wt %) of the triblock copolymerrendered the particles mucus penetrating, whereas mucoadhesion wasgenerally observed with particles having weight percentages ofhydrophilic blocks below this limit. In some embodiments, thehydrophilic blocks of a triblock copolymer constitute at least about 15wt %, at least about 20 wt %, at least about 25 wt %, at least about 30wt %, at least about 35 wt %, at least about 40 wt %, at least about 45wt %, at least about 50 wt %, at least about 55 wt %, at least about 60wt %, at least about 65 wt %, or at least about 70 wt % of the triblockcopolymer. In some embodiments, the hydrophilic blocks of a triblockcopolymer constitute less than or equal to about 90 wt %, less than orequal to about 80 wt %, less than or equal to about 60 wt %, less thanor equal to about 50 wt %, or less than or equal to about 40 wt % of thetriblock copolymer. Combinations of the above-referenced ranges are alsopossible (e.g., at least about 30 wt % and less than or equal to about80 wt %). Other ranges are also possible.

In some embodiments, the molecular weight of a hydrophilic block (e.g.,a PEG (or PEO) block of the triblock copolymer PEG-PPO-PEG, where a PEGblock may be interchanged with a PEO block) may be at least about 0.05kDa, at least about 0.1 kDa, at least about 0.2 kDa, at least about 0.3kDa, at least about 0.4 kDa, at least about 0.5 kDa, at least about 1kDa, at least about 2 kDa, at least about 3 kDa, at least about 4 kDa,at least about 5 kDa, or at least about 6 kDa, at least about 8 kDa, atleast about 10 kDa, at least about 20 kDa, or at least about 50 kDa. Incertain embodiments, the molecular weight of a hydrophilic block may beless than or equal to about 100 kDa, less than or equal to about 80 kDa,less than or equal to about 50 kDa, less than or equal to about 20 kDa,less than or equal to about 15 kDa, less than or equal to about 10 kDa,less than or equal to about 9 kDa, less than or equal to about 8 kDa,less than or equal to about 7 kDa, less than or equal to about 6 kDa,less than or equal to about 5 kDa, less than or equal to about 3 kDa,less than or equal to about 2 kDa, or less than or equal to about 1 kDa.Combinations of the above-mentioned ranges are also possible (e.g., atleast about 0.1 kDa and less than or equal to about 3 kDa). Other rangesare also possible. In embodiments in which two hydrophilic blocks flanka hydrophobic block, the molecular weights of the two hydrophilic blocksmay be substantially the same or different.

In certain embodiments, the polymer of a surface-altering agent includesa polyether portion. In certain embodiments, the polymer includes apolyalkylether portion. In certain embodiments, the polymer includespolyethylene glycol tails. In certain embodiments, the polymer includesa polypropylene glycol central portion. In certain embodiments, thepolymer includes polybutylene glycol as the central portion. In certainembodiments, the polymer includes polypentylene glycol as the centralportion. In certain embodiments, the polymer includes polyhexyleneglycol as the central portion. In certain embodiments, the polymer is atriblock copolymer of one of the polymers described herein. As disclosedherein, any recitation of PEG may be replaced with polyethylene oxide(PEO), and any recitation of PEO may be replaced with PEG.

In certain embodiments, the polymer is a triblock copolymer of apolyalkyl ether (e.g., polyethylene glycol, polypropylene glycol) andanother polymer. In certain embodiments, the polymer is a triblockcopolymer of a polyalkyl ether and another polyalkyl ether. In certainembodiments, the polymer is a triblock copolymer of polyethylene glycoland another polyalkyl ether. In certain embodiments, the polymer is atriblock copolymer of polypropylene glycol and another polyalkyl ether.In certain embodiments, the polymer is a triblock copolymer with atleast one unit of polyalkyl ether. In certain embodiments, the polymeris a triblock copolymer of two different polyalkyl ethers. In certainembodiments, the polymer is a triblock copolymer including apolyethylene glycol unit. In certain embodiments, the polymer is atriblock copolymer including a polypropylene glycol unit. In certainembodiments, the polymer is a triblock copolymer of a more hydrophobicunit flanked by two more hydrophilic units. In certain embodiments, thehydrophilic units are the same type of polymer. In certain embodiments,the polymer includes a polypropylene glycol unit flanked by two morehydrophilic units. In certain embodiments, the polymer includes twopolyethylene glycol units flanking a more hydrophobic unit. In certainembodiments, the polymer is a triblock copolymer with a polypropyleneglycol unit flanked by two polyethylene glycol units. The molecularweights of the two blocks flanking the central block may besubstantially the same or different.

In certain embodiments, the polymer is of the formula:

wherein n is an integer between 2 and 1140, inclusive; and m is aninteger between 2 and 1730, inclusive. In certain embodiments, n is aninteger between 10 and 170, inclusive. In certain embodiments, m is aninteger between 5 and 70 inclusive. In certain embodiments, n is atleast 2 times m, 3 times m, or 4 times m.

In certain embodiments, the coating includes a surface-altering agentcomprising a (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (hereinafter“PEG-PPO-PEG triblock copolymer”). As described herein, the PEG blocksmay be interchanged with PEO blocks in some embodiments. The molecularweights of the PEG (or PEO) and PPO segments of the PEG-PPO-PEG triblockcopolymer may be selected so as to reduce the mucoadhesion of theparticle, as described herein. Without wishing to be bound by theory, aparticle having a coating comprising a PEG-PPO-PEG triblock copolymermay have reduced mucoadhesion as compared to a control particle due to,at least in part, the display of a plurality of PEG (or PEO) segments onthe particle surface. The PPO segment may be adhered to the core surface(e.g., in the case of the core surface being hydrophobic), thus allowingfor a strong association between the core and the triblock copolymer. Insome cases, the PEG-PPO-PEG triblock copolymer is associated with thecore through non-covalent interactions. For purposes of comparison, thecontrol particle may be, for example, a carboxylate-modified polystyreneparticle of similar size as the coated particle in question.

In certain embodiments, a surface-altering agent includes a polymercomprising a poloxamer, having the trade name Pluronic®. Pluronic®polymers that may be useful in the embodiments described herein include,but are not limited to, F127, F38, F108, F68, F77, F87, F88, F98, L101,L121, L31, L35, L43, L44, L61, L62, L64, L81, L92, N3, P103, P104, P105,P123, P65, P84, and P85.

Examples of molecular weights of certain Pluronic® molecules are shownin Table 1.

TABLE 1 Molecular Weights of Pluronic ® molecules Pluronic ® Average MWMW PPO PEO wt % MW PEO L31 1000 900 10 100 L44 2000 1200 40 800 L81 26672400 10 267 L101 3333 3000 10 333 P65 3600 1800 50 1800 L121 4000 360010 400 P103 4286 3000 30 1286 F38 4500 900 80 3600 P123 5143 3600 301543 P105 6000 3000 50 3000 F87 8000 2400 70 5600 F68 9000 1800 80 7200F127 12000 3600 70 8400 P123 5750 4030 30 1730

Although other ranges may be possible and useful in certain embodimentsdescribed herein, in some embodiments, the hydrophobic block of thePEG-PPO-PEG triblock copolymer has one of the molecular weightsdescribed above (e.g., at least about 3 kDa and less than or equal toabout 15 kDa), and the combined hydrophilic blocks have a weightpercentage with respect to the polymer in one of the ranges describedabove (e.g., at least about 15 wt %, at least about 20 wt %, at leastabout 25 wt %, or at least about 30 wt %, and less than or equal toabout 80 wt %). Certain Pluronic® polymers that fall within thesecriteria include, for example, F127, F108, P105 and P103. Surprisingly,and as described in more detail in the Examples, it was found that theseparticular Pluronic® polymers rendered particles mucus penetrating morethan other Pluronic® polymers tested that did not fall within thiscriteria. Additionally, other agents that did not render particles mucuspenetrating included certain polymers such as polyvinylpyrrolidones(PVP/Kollidon), polyvinyl alcohol-polyethylene glycol graft-copolymer(Kollicoat IR), hydroxypropyl methylcellulose (Methocel); oligomers suchas Tween 20, Tween 80, solutol HS 15, Triton X100, tyloxapol, cremophorRH 40; small molecules such as Span 20, Span 80, octyl glucoside,cetytrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS).

Although much of the description herein involves coatings comprising ahydrophilic block—hydrophobic block—hydrophilic block configuration(e.g., a PEG-PPO-PEG triblock copolymer), it should be appreciated thatthe coatings are not limited to this configuration and that otherconfigurations and materials are possible. For example, a particle mayinclude more than one coating (e.g., at least two, three, four, five, ormore coatings), and each coating need not be formed of or comprise amucus penetrating material. In some cases, an intermediate coating(i.e., a coating between the core surface and an outer coating) mayinclude a polymer that facilitates attachment of an outer coating to thecore surface. In many embodiments, an outer coating of a particleincludes a polymer comprising a material that facilitates the transportof the particle through mucus.

As such, a coating (e.g., an inner coating, an intermediate coating,and/or an outer coating) may include any suitable polymer. In somecases, the polymer may be biocompatible and/or biodegradable. In somecases, the polymeric material may comprise more than one type of polymer(e.g., at least two, three, four, five, or more, polymers). In somecases, a polymer may be a random copolymer or a block copolymer (e.g., adiblock copolymer, a triblock copolymer) as described herein.

Non-limiting examples of suitable polymers may include polyamines,polyethers, polyamides, polyesters, polycarbamates, polyureas,polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes,polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates,polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.Non-limiting examples of specific polymers include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (jointlyreferred to herein as “polyacrylic acids”), and copolymers and mixturesthereof, polydioxanone and its copolymers, polyhydroxyalkanoates,polypropylene fumarate), polyoxymethylene, poloxamers,poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone.

The molecular weight of a polymer may vary. In some embodiments, themolecular weight may be at least about 0.5 kDa, at least about 1 kDa, atleast about 2 kDa, at least about 3 kDa, at least about 4 kDa, at leastabout 5 kDa, at least about 6 kDa, at least about 8 kDa, at least about10 kDa, at least about 12 kDa, at least about 15 kDa, at least about 20kDa, at least about 30 kDa, at least about 40 kDa, or at least about 50kDa. In some embodiments, the molecular weight may be less than or equalto about 50 kDa, less than or equal to about 40 kDa, less than or equalto about 30 kDa, less than or equal to about 20 kDa, less than or equalto about 12 kDa, less than or equal to about 10 kDa, less than or equalto about 8 kDa, less than or equal to about 6 kDa, less than or equal toabout 5 kDa, or less than or equal to about 4 kDa. Combinations of theabove-referenced ranges are possible (e.g., a molecular weight of atleast about 2 kDa and less than or equal to about 15 kDa). Other rangesare also possible. The molecular weight may be determined using anyknown technique such as light-scattering and gel permeationchromatography. Other methods are known in the art.

In certain embodiments, the polymer is biocompatible, i.e., the polymerdoes not typically induce an adverse response when inserted or injectedinto a living subject; for example, it does not include significantinflammation and/or acute rejection of the polymer by the immune system,for instance, via a T-cell-mediated response. It will be recognized, ofcourse, that “biocompatibility” is a relative term, and some degree ofimmune response is to be expected even for polymers that are highlycompatible with living tissue. However, as used herein,“biocompatibility” refers to the acute rejection of material by at leasta portion of the immune system, i.e., a non-biocompatible materialimplanted into a subject provokes an immune response in the subject thatis severe enough such that the rejection of the material by the immunesystem cannot be adequately controlled, and often is of a degree suchthat the material must be removed from the subject. One simple test todetermine biocompatibility is to expose a polymer to cells in vitro;biocompatible polymers are polymers that typically does not result insignificant cell death at moderate concentrations, e.g., atconcentrations of about 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. In some embodiments, asubstance is “biocompatible” if its addition to cells in vitro resultsin less than or equal to 20% cell death, and their administration invivo does not induce unwanted inflammation or other such adverseeffects.

In certain embodiments, a biocompatible polymer may be biodegradable,i.e., the polymer is able to degrade, chemically and/or biologically(e.g., by the cellular machinery or by hydrolysis), within aphysiological environment, such as within the body or when introduced tocells. For instance, the polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), and/orthe polymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer is degraded intomonomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymer maybe biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymer may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (i.e., fewer than about 20% of the cells are killed when thecomponents are added to cells in vitro). For example, polylactide may behydrolyzed to form lactic acid, polyglycolide may be hydrolyzed to formglycolic acid, etc.).

Examples of biodegradable polymers include, but are not limited to,poly(ethylene glycol)-poly(propylene oxide)-poly(ethylene glycol)triblock copolymers, poly(lactide) (or poly(lactic acid)),poly(glycolide) (or poly(glycolic acid)), poly(orthoesters),poly(caprolactones), polylysine, poly(ethylene imine), poly(acrylicacid), poly(urethanes), poly(anhydrides), poly(esters),poly(trimethylene carbonate), poly(ethyleneimine), poly(acrylic acid),poly(urethane), poly(beta amino esters) or the like, and copolymers orderivatives of these and/or other polymers, for example,poly(lactide-co-glycolide) (PLGA).

In certain embodiments, a polymer may biodegrade within a period that isacceptable in the desired application. In certain embodiments, such asin vivo therapy, such degradation occurs in a period usually less thanabout five years, one year, six months, three months, one month, fifteendays, five days, three days, or even one day or less (e.g., 1-4 hours,4-8 hours, 4-24 hours, 1-24 hours) on exposure to a physiologicalsolution with a pH between 6 and 8 having a temperature of between 25and 37° C. In other embodiments, the polymer degrades in a period ofbetween about one hour and several weeks, depending on the desiredapplication.

Although coatings and particles described herein may include polymers,in some embodiments, the particles described herein comprise ahydrophobic material that is not a polymer (e.g., a non-polymer) and isnot a pharmaceutical agent. For example, all or portions of a particlemay be coated with a passivating layer in some embodiments. Non-limitingexamples of non-polymeric materials may include certain metals, waxes,and organic materials (e.g., organic silanes, perfluorinated orfluorinated organic materials).

Particles with Reduced Mucoadhesion

As described herein, in some embodiments, a method involves identifyinga material such as a particle to which it is desired that itsmucoadhesiveness be reduced. Materials in need of increased diffusivitythrough mucus may be, for example, hydrophobic, have many hydrogen bonddonors or acceptors, and/or may be highly charged. In some cases, thematerial may include a crystalline or amorphous solid material. Thematerial, which may serve as a core, may be coated with a suitablepolymer described herein, thereby forming a particle with a plurality ofsurface-altering moieties on the surface, resulting in reducedmucoadhesion. Particles herein described as having reduced mucoadhesionmay alternatively be characterized as having increased transport throughmucus, being mobile in mucus, or mucus-penetrating (i.e.,mucus-penetrating particles), meaning that the particles are transportedthrough mucus faster than a (negative) control particle. The (negative)control particle may be a particle that is known to be mucoadhesive,e.g., an unmodified particle or core that is not coated with a coatingdescribed herein, such as a 200 nm carboxylated polystyrene particle.

In certain embodiments, methods herein include preparing apharmaceutical composition or formulation of the modified substance,e.g., in a formulation adapted for delivery (e.g., topical delivery) tomucus or a mucosal surface of a subject. The pharmaceutical compositionwith surface-altering moieties may be delivered to the mucosal surfaceof a subject, may pass through the mucosal barrier in the subject,and/or prolonged retention and/or increased uniform distribution of theparticles at mucosal surfaces, e.g., due to reduced mucoadhesion. Aswill be known by those of ordinary skill in the art, mucus is aviscoelastic and adhesive substance that traps most foreign particles.Trapped particles are not able to reach the underlying epithelium and/orare quickly eliminated by mucus clearance mechanisms. For a particle toreach the underlying epithelium and/or for a particle to have prolongedretention in the mucosal tissue, the particle must quickly penetratemucus secretions and/or avoid the mucus clearance mechanisms. If aparticle does not adhere substantially to the mucus, the particle may beable to diffuse in the interstitial fluids between mucin fibers andreach the underlying epithelium and/or not be eliminated by the mucusclearance mechanisms. Accordingly, modifying mucoadhesive materials,(e.g., pharmaceutical agents that are hydrophobic) with a material toreduce the mucoadhesion of the particle may allow for efficient deliveryof the particles to the underlying epithelium and/or prolonged retentionat mucosal surfaces.

Furthermore, in some embodiments, the particles described herein havingreduced mucoadhesion facilitate better distribution of the particles ata tissue surface, and/or have a prolonged presence at the tissuesurface, compared to particles that are more mucoadhesive. For example,in some cases a luminal space such as the gastrointestinal tract issurrounded by a mucus-coated surface. Mucoadhesive particles deliveredto such a space are typically removed from the luminal space and fromthe mucus-coated surface by the body's natural clearance mechanisms. Theparticles described herein with reduced mucoadhesion may remain in theluminal space for relatively longer periods compared to the mucoadhesiveparticles. This prolonged presence may prevent or reduce clearance ofthe particles, and/or may allow for better distribution of the particleson the tissue surface. The prolonged presence may also affect theparticle transport through the luminal space, e.g., the particles maydistribute into the mucus layer and may reach the underlying epithelium.

In certain embodiments, a material (e.g., a core) coated with a polymerdescribed herein may pass through mucus or a mucosal barrier in asubject, and/or exhibit prolonged retention and/or increase uniformdistribution of the particles at mucosal surfaces, e.g., such substancesare cleared more slowly (e.g., at least 2 times, 5 times, 10 times, oreven at least 20 times more slowly) from a subject's body as compared toa (negative) control particle. The (negative) control particle may be aparticle that is known to be mucoadhesive, e.g., an unmodified particleor core that is not coated with a coating described herein, such as a200 nm carboxylated polystyrene particle.

In certain embodiments, a particle described herein has certain arelative velocity, <V_(mean)>_(rel), which is defined as follows:

$\begin{matrix}{\left\langle V_{mean} \right\rangle_{rel} = \frac{\left\langle V_{mean} \right\rangle_{Sample} - \left\langle V_{mean} \right\rangle_{{Negative}\mspace{20mu}{control}}}{\left\langle V_{mean} \right\rangle_{{Positive}\mspace{14mu}{contol}} - \left\langle V_{mean} \right\rangle_{{Negative}\mspace{14mu}{control}}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$where <V_(mean)> is the ensemble average trajectory-mean velocity,V_(mean) is the velocity of an individual particle averaged over itstrajectory, the sample is the particle of interest, the negative controlis a 200 nm carboxylated polystyrene particle, and the positive controlis a 200 nm polystyrene particle densely PEGylated with 2 kDa-5 kDa PEG.

The relative velocity can be measured by a multiple particle trackingtechnique. For instance, a fluorescent microscope equipped with a CCDcamera can be used to capture 15 s movies at a temporal resolution of66.7 ms (15 frames/s) under 100× magnification from several areas withineach sample for each type of particles: sample, negative control, andpositive control. The sample, negative and positive controls may befluorescent particles to observe tracking. Alternatively non-fluorescentparticles may be coated with a fluorescent molecule, a fluorescentlytagged surface agent or a fluorescently tagged polymer. An advancedimage processing software (e.g., Image Pro or MetaMorph) can be used tomeasure individual trajectories of multiple particles over a time-scaleof at least 3.335 s (50 frames).

In some embodiments, a particle described herein has a relative velocityof greater than about 0.3, greater than about 0.4, greater than about0.5, greater than about 0.6, greater than about 0.7, greater than about0.8, greater than about 0.9, greater than about 1.0, greater than about1.1, greater than about 1.2, greater than about 1.3, greater than about1.4, greater than about 1.5, greater than about 1.6, greater than about1.7, greater than about 1.8, greater than about 1.9 or greater thanabout 2.0 in mucus. In some embodiments, a particle described herein hasa relative velocity of less than or equal to about 10.0, less than orequal to about 8.0, less than or equal to about 6.0, less than or equalto about 4.0, less than or equal to about 3.0, less than or equal toabout 2.0, less than or equal to about 1.9, less than or equal to about1.8, less than or equal to about 1.7, less than or equal to about 1.6,less than or equal to about 1.5, less than or equal to about 1.4, lessthan or equal to about 1.3, less than or equal to about 1.2, less thanor equal to about 1.1, less than or equal to about 1.0, less than orequal to about 0.9, less than or equal to about 0.8, or less than orequal to about 1.7 in mucus. Combinations of the above-noted ranges arepossible (e.g., a relative velocity of greater than about 0.5 and lessthan or equal to about 6.0). Other ranges are also possible. The mucusmay be, for example, human cervicovaginal mucus.

In certain embodiments, a particle described herein can diffuse throughmucus or a mucosal barrier at a greater rate or diffusivity than acontrol particle or a corresponding particle (e.g., a correspondingparticle that is unmodified and/or is not coated with a coatingdescribed herein). In some cases, a particle described herein may passthrough mucus or a mucosal barrier at a rate of diffusivity that is atleast about 10 times, 20 times, 30 times, 50 times, 100 times, 200times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times, ormore, higher than a control particle or a corresponding particle. Insome cases, a particle described herein may pass through mucus or amucosal barrier at a rate of diffusivity that is less than or equal toabout 10000 times higher, less than or equal to about 5000 times higher,less than or equal to about 2000 times higher, less than or equal toabout 1000 times higher, less than or equal to about 500 times higher,less than or equal to about 200 times higher, less than or equal toabout 100 times higher, less than or equal to about 50 times higher,less than or equal to about 30 times higher, less than or equal to about20 times higher, or less than or equal to about 10 times higher than acontrol particle or a corresponding particle. Combinations of theabove-referenced ranges are also possible (e.g., at least about 10 timesand less than or equal to about 1000 times higher than a controlparticle or a corresponding particle). Other ranges are also possible.

For the purposes of the comparisons described herein, the correspondingparticle may be approximately the same size, shape, and/or density asthe test particle but lacking the coating that makes the test particlemobile in mucus. In some cases, the measurement is based on a time scaleof about 1 second, or about 0.5 second, or about 2 seconds, or about 5seconds, or about 10 seconds. Those of ordinary skill in the art will beaware of methods for determining the geometric mean square displacementand rate of diffusivity.

In addition, a particle described herein may pass through mucus or amucosal barrier with a geometric mean squared displacement that is atleast about 10 times, 20 times, 30 times, 50 times, 100 times, 200times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times, ormore, higher than a corresponding particle or control particle. In somecases, a particle described herein may pass through mucus or a mucosalbarrier with a geometric mean squared displacement that is less than orequal to about 10000 times higher, less than or equal to about 5000times higher, less than or equal to about 2000 times higher, less thanor equal to about 1000 times higher, less than or equal to about 500times higher, less than or equal to about 200 times higher, less than orequal to about 100 times higher, less than or equal to about 50 timeshigher, less than or equal to about 30 times higher, less than or equalto about 20 times higher, or less than or equal to about 10 times higherthan a control particle or a corresponding particle. Combinations of theabove-referenced ranges are also possible (e.g., at least about 10 timesand less than or equal to about 1000 times higher than a controlparticle or a corresponding particle). Other ranges are also possible.

In some embodiments, a particle described herein diffuses through amucosal barrier at a rate approaching the rate or diffusivity at whichsaid particles can diffuse through water. In some cases, a particledescribed herein may pass through a mucosal barrier at a rate ordiffusivity that is less than or equal to about ½, less than or equal toabout ¼, less than or equal to about ⅛, less than or equal to about1/16, less than or equal to about 1/32, less than or equal to about1/50, less than or equal to about 1/100, less than or equal to about1/200, less than or equal to about 1/300, less than or equal to about1/400, less than or equal to about 1/500, less than or equal to about1/600, less than or equal to about 1/700, less than or equal to about1/800, less than or equal to about 1/900, less than or equal to about1/1000, less than or equal to about 1/2000, less than or equal to about1/5000, less than or equal to about 1/10,000 the diffusivity that theparticle diffuse through water under identical conditions. In somecases, a particle described herein may pass through a mucosal barrier ata rate or diffusivity that is greater than about 1/10,000, greater thanabout 1/5000, greater than about 1/2000, greater than about 1/1000,greater than about 1/900, greater than about 1/800, greater than about1/700, greater than about 1/600, greater than about 1/500, greater thanabout 1/400, greater than about 1/300, greater than about 1/200, greaterthan about 1/100, greater than about 1/50, greater than about 1/32,greater than about 1/16, greater than about ⅛, greater than about ¼, orgreater than about ½ the diffusivity that the particle diffuse throughwater under identical conditions. Combinations of the above-referencedranges are also possible (e.g., greater than about 1/5000 and less than1/500 the diffusivity that the particle diffuse through water underidentical conditions). Other ranges are also possible. The measurementmay be based on a time scale of about 1 second, or about 0.5 second, orabout 2 seconds, or about 5 seconds, or about 10 seconds.

In a particular embodiment, a particle described herein may diffusethrough human cervicovaginal mucus at a diffusivity that is less thanabout 1/500 the diffusivity that the particle diffuses through water. Insome cases, the measurement is based on a time scale of about 1 second,or about 0.5 second, or about 2 seconds, or about 5 seconds, or about 10seconds.

In certain embodiments, the present invention provides particles thattravel through mucus, such as human cervicovaginal mucus, at certainabsolute diffusivities. For example, the particles of described hereinmay travel at diffusivities of at least about 1×10⁻⁴ μm/s, 2×10⁻⁴ μm/s,5×10⁻⁴ μm/s, 1×10⁻³ μm/s, 2×10⁻³ μm/s, 5×10⁻³ μm/s, 1×10⁻² μm/s, 2×10⁻²μm/s, 4×10⁻² μm/s, 5×10⁻² m/s, 6×10⁻² μm/s, 8×10⁻² μm/s, 1×10⁻¹ μm/s,2×10⁻¹ μm/s, 5×10⁻¹ μm/s, 1 μm/s, or 2 μm/s. In some cases, theparticles may travel at diffusivities of less than or equal to about 2μm/s, less than or equal to about 1 μm/s, less than or equal to about5×10⁻¹ μm/s, less than or equal to about 2×10⁻¹ μm/s, less than or equalto about 1×10⁻¹ μm/s, less than or equal to about 8×10⁻² μm/s, less thanor equal to about 6×10⁻² μm/s, less than or equal to about 5×10⁻² μm/s,less than or equal to about 4×10⁻² μm/s, less than or equal to about2×10⁻² μm/s, less than or equal to about 1×10⁻² μm/s, less than or equalto about 5×10⁻³ μm/s, less than or equal to about 2×10⁻³ μm/s, less thanor equal to about 1×10⁻³ μm/s, less than or equal to about 5×10⁻⁴ μm/s,less than or equal to about 2×10⁻⁴ μm/s, or less than or equal to about1×10⁻⁴ μm/s. Combinations of the above-referenced ranges are alsopossible (e.g., greater than about 2×10⁻⁴ am/s and less than or equal toabout 1×10¹ μm/s). Other ranges are also possible. In some cases, themeasurement is based on a time scale of about 1 second, or about 0.5second, or about 2 seconds, or about 5 seconds, or about 10 seconds.

It should be appreciated that while many of the mobilities (e.g.,relative velocities, diffusivities) described here may be measured inhuman cervicovaginal mucus, they may be measured in other types of mucusas well.

In certain embodiments, a particle described herein comprisessurface-altering moieties at a given density. The surface-alteringmoieties may be the portions of a surface-altering agent that are, forexample, exposed to the solvent containing the particle. As an example,the PEG segments may be surface-altering moieties of thesurface-altering agent PEG-PPO-PEG. In some cases, the surface-alteringmoieties and/or surface-altering agents are present at a density of atleast about 0.001 units or molecules per nm², at least about 0.002, atleast about 0.005, at least about 0.01, at least about 0.02, at leastabout 0.05, at least about 0.1, at least about 0.2, at least about 0.5,at least about 1, at least about 2, at least about 5, at least about 10,at least about 20, at least about 50, at least about 100 units ormolecules per nm², or more units or molecules per nm². In some cases,the surface-altering moieties and/or surface-altering agents are presentat a density of less than or equal to about 100 units or molecules pernm², less than or equal to about 50, less than or equal to about 20,less than or equal to about 10, less than or equal to about 5, less thanor equal to about 2, less than or equal to about 1, less than or equalto about 0.5, less than or equal to about 0.2, less than or equal toabout 0.1, less than or equal to about 0.05, less than or equal to about0.02, or less than or equal to about 0.01 units or molecules per nm².Combinations of the above-referenced ranges are possible (e.g., adensity of at least about 0.01 and less than or equal to about 1 unitsor molecules per nm²). Other ranges are also possible.

Those of ordinary skill in the art will be aware of methods to estimatethe average density of surface-altering moieties (see, for example, S.J. Budijono et al., Colloids and Surfaces A: Physicochem. Eng. Aspects360 (2010) 105-110; Joshi, et al. Anal. Chim. Acta 104 (1979) 153-160,each of which is incorporated herein by reference). For example, asdescribed herein, the average density of surface-altering moieties canbe determined using HPLC quantitation and DLS analysis. A suspension ofparticles for which surface density determination is of interest isfirst sized using DLS: a small volume is diluted to an appropriateconcentration (˜100 μg/mL, for example), and the z-average diameter istaken as a representative measurement of particle size. The remainingsuspension is then divided into two aliquots. Using HPLC, the firstaliquot is assayed for the total concentration of core material and forthe total concentration of surface-altering moiety. Again using HPLC thesecond aliquot is assayed for the concentration of free or unboundsurface-altering moiety. In order to get only the free or unboundsurface-altering moiety from the second aliquot, the particles, andtherefore any bound surface-altering moiety, are removed byultracentrifugation. By subtracting the concentration of the unboundsurface-altering moiety from the total concentration of surface-alteringmoiety, the concentration of bound surface-altering moiety can bedetermined. Since the total concentration of core material was alsodetermined from the first aliquot, the mass ratio between the corematerial and the surface-altering moiety can be determined. Using themolecular weight of the surface-altering moiety the number ofsurface-altering moiety to mass of core material can be calculated. Toturn this number into a surface density measurement, the surface areaper mass of core material needs to be calculated. The volume of theparticle is approximated as that of a sphere with the diameter obtainedfrom DLS allowing for the calculation of the surface area per mass ofcore material. In this way the number of surface-altering moieties persurface area can be determined.

Density may also be measured by quantifying or estimating the corematerial surface area by methods such as electron microscopy, lightscattering, or surface interaction measurements and then quantifying theadhered surface agent(s) by methods such as liquid chromatography ormass spectrometry. (See, for example, Wang et al., Angew Chem Int EdEngl, 2008, 47(50), 9726-9, which is incorporated herein by reference).

In certain embodiments, the particles described herein comprisesurface-altering moieties and/or agents that affect the zeta-potentialof the particle. The zeta potential of the coated particle may be, forexample, at least about −100 mV, at least about −75 mV, at least about−50 mV, at least about −40 mV, at least about −30 mV, at least about −20mV, at least about −10 mV, at least about −5 mV, at least about 5 mV, atleast about 10 mV, at least about 20 mV, at least about 30 mV, at leastabout 40 mV, at least about 50 mV, at least about 75 mV, or at leastabout 100 mV. Combinations of the above-referenced ranges are possible(e.g., a zeta-potential of at least about −50 mV and less than or equalto about 50 mV). Other ranges are also possible.

The coated particles described herein may have any suitable shape and/orsize. In some embodiments, a coated particle has a shape substantiallysimilar to the shape of the core. In some cases, a coated particledescribed herein may be a nanoparticle, i.e., the particle has acharacteristic dimension of less than about 1 micrometer, where thecharacteristic dimension of the particle is the diameter of a perfectsphere having the same volume as the particle. In other embodiments,larger sizes are possible (e.g., about 1-10 microns). A plurality ofparticles, in some embodiments, may also be characterized by an averagesize (e.g., an average largest cross-sectional dimension, or an averagesmallest cross-sectional dimension for the plurality of particles). Aplurality of particles may have an average size of, for example, lessthan or equal to about 10 μm, less than or equal to about 5 μm, lessthan or equal to about 1 μm, less than or equal to about 800 nm, lessthan or equal to about 700 nm, less than or equal to about 500 nm, lessthan or equal to 400 nm, less than or equal to 300 nm, less than orequal to about 200 nm, less than or equal to about 100 nm, less than orequal to about 75 nm, less than or equal to about 50 nm, less than orequal to about 40 nm, less than or equal to about 35 nm, less than orequal to about 30 nm, less than or equal to about 25 nm, less than orequal to about 20 nm, less than or equal to about 15 nm, or less than orequal to about 5 nm. In some cases, a plurality of particles may have anaverage size of, for example, at least about 5 nm, at least about 20 nm,at least about 50 nm, at least about 100 nm, at least about 200 nm, atleast about 300 nm, at least about 400 nm, at least about 500 nm, atleast about 1 μm, at least or at least about 5 μm. Combinations of theabove-referenced ranges are also possible (e.g., an average size of atleast about 50 nm and less than about 500 nm). Other ranges are alsopossible. In some embodiments, the sizes of the cores formed by aprocess described herein have a Gaussian-type distribution.

Pharmaceutical Agents

In some embodiments, a coated particle comprises at least onepharmaceutical agent. The pharmaceutical agent may be present in thecore of the particle and/or present in a coating of the particle (e.g.,dispersed throughout the core and/or coating). In some cases, apharmaceutical agent may be disposed on the surface of the particle(e.g., on an outer surface of a coating, the inner surface of a coating,on a surface of the core). The pharmaceutical agent may be containedwithin a particle and/or disposed in a portion of the particle usingcommonly known techniques (e.g., by coating, adsorption, covalentlinkage, or other process). In some cases, the pharmaceutical agent maybe present in the core of the particle prior to or during coating of theparticle. In some cases, the pharmaceutical agent is present during theformation of the core of the particle, as described herein.

Non-limiting examples of pharmaceutical agents include imaging agents,diagnostic agents, therapeutic agents, agents with a detectable label,nucleic acids, nucleic acid analogs, small molecules, peptidomimetics,proteins, peptides, lipids, vaccines, viral vectors, virus, andsurfactants.

In some embodiments, a pharmaceutical agent contained in a particledescribed herein has a therapeutic, diagnostic, or imaging effect in amucosal tissue to be targeted. Non-limiting examples of mucosal tissuesinclude oral (e.g., including the buccal and esophagal membranes andtonsil surface), ophthalmic, gastrointestinal (e.g., including stomach,small intestine, large intestine, colon, rectum), nasal, respiratory(e.g., including nasal, pharyngeal, tracheal and bronchial membranes),and genital (e.g., including vaginal, cervical and urethral membranes)tissues.

Any suitable number of pharmaceutical agents may be present in aparticle described herein. For example, at least 1, at least 2, at least3, at least 4, at least 5, or more, but generally less than 10,pharmaceutical agents may be present in a particle described herein.

A number of drugs that are mucoadhesive are known in the art and may beused as pharmaceutical agents in the particles described herein (see,for example, Khanvilkar K, et al. Adv Drug Del Rev 48:173-193, 2001;Bhat P G et al. J Pharm Sci 85:624-30, 1996). Additional non-limitingexamples of pharmaceutical agents include imaging and diagnostic agents(such as radioopaque agents, labeled antibodies, labeled nucleic acidprobes, dyes, such as colored or fluorescent dyes, etc.) and adjuvants(radiosensitizers, transfection-enhancing agents, chemotactic agents andchemoattractants, peptides that modulate cell adhesion and/or cellmobility, cell permeabilizing agents, vaccine potentiators, inhibitorsof multidrug resistance and/or efflux pumps, etc.).

Additional non-limiting examples of pharmaceutical agents includealoxiprin, auranofin, azapropazone, benorylate, diflunisal, etodolac,fenbufen, fenoprofen calcim, flurbiprofen, furosemide, ibuprofen,indomethacin, ketoprofen, loteprednol etabonate, meclofenamic acid,mefenamic acid, nabumetone, naproxen, oxyphenbutazone, phenylbutazone,piroxicam, sulindac, albendazole, bephenium hydroxynaphthoate,cambendazole, dichlorophen, ivermectin, mebendazole, oxamniquine,oxfendazole, oxantel embonate, praziquantel, pyrantel embonate,thiabendazole, amiodarone HCl, disopyramide, flecainide acetate,quinidine sulphate. Anti-bacterial agents: benethamine penicillin,cinoxacin, ciprofloxacin HCl, clarithromycin, clofazimine, cloxacillin,demeclocycline, doxycycline, erythromycin, ethionamide, imipenem,nalidixic acid, nitrofurantoin, rifampicin, spiramycin, sulphabenzamide,sulphadoxine, sulphamerazine, sulphacetamide, sulphadiazine,sulphafurazole, sulphamethoxazole, sulphapyridine, tetracycline,trimethoprim, dicoumarol, dipyridamole, nicoumalone, phenindione,amoxapine, maprotiline HCl, mianserin HCL, nortriptyline HCl, trazodoneHCL, trimipramine maleate, acetohexamide, chlorpropamide, glibenclamide,gliclazide, glipizide, tolazamide, tolbutamide, beclamide,carbamazepine, clonazepam, ethotoin, methoin, methsuximide,methylphenobarbitone, oxcarbazepine, paramethadione, phenacemide,phenobarbitone, phenytoin, phensuximide, primidone, sulthiame, valproicacid, amphotericin, butoconazole nitrate, clotrimazole, econazolenitrate, fluconazole, flucytosine, griseofulvin, itraconazole,ketoconazole, miconazole, natamycin, nystatin, sulconazole nitrate,terbinafine HCl, terconazole, tioconazole, undecenoic acid, allopurinol,probenecid, sulphin-pyrazone, amlodipine, benidipine, darodipine,dilitazem HCl, diazoxide, felodipine, guanabenz acetate, isradipine,minoxidil, nicardipine HCl, nifedipine, nimodipine, phenoxybenzamineHCl, prazosin HCL, reserpine, terazosin HCL, amodiaquine, chloroquine,chlorproguanil HCl, halofantrine HCl, mefloquine HCl, roguanil HCl,pyrimethamine, quinine sulphate, dihydroergotamine mesylate, ergotaminetartrate, methysergide maleate, pizotifen maleate, sumatriptansuccinate, atropine, benzhexol HCl, biperiden, ethopropazine HCl,hyoscyamine, mepenzolate bromide, oxyphencylcimine HCl, tropicamide,aminoglutethimide, amsacrine, azathioprine, busulphan, chlorambucil,cyclosporin, dacarbazine, estramustine, etoposide, lomustine, melphalan,mercaptopurine, methotrexate, mitomycin, mitotane, mitozantrone,procarbazine HCl, tamoxifen citrate, testolactone, benznidazole,clioquinol, decoquinate, diiodohydroxyquinoline, diloxanide furoate,dinitolmide, furzolidone, metronidazole, nimorazole, nitrofurazone,ornidazole, tinidazole, carbimazole, propylthiouracil, alprazolam,amylobarbitone, barbitone, bentazepam, bromazepam, bromperidol,brotizolam, butobarbitone, carbromal, chlordiazepoxide, chlormethiazole,chlorpromazine, clobazam, clotiazepam, clozapine, diazepam, droperidol,ethinamate, flunanisone, flunitrazepam, fluopromazine, flupenthixoldecanoate, fluphenazine decanoate, flurazepam, haloperidol, lorazepam,lormetazepam, medazepam, meprobamate, methaqualone, midazolam,nitrazepam, oxazepam, pentobarbitone, perphenazine pimozide,prochlorperazine, sulpiride, temazepam, thioridazine, triazolam,zopiclone, acebutolol, alprenolol, atenolol, labetalol, metoprolol,nadolol, oxprenolol, pindolol, propranolol, amrinone, digitoxin,digoxin, enoximone, lanatoside C, medigoxin, beclomethasone,betamethasone, budesonide, cortisone acetate, desoxymethasone,dexamethasone, fludrocortisone acetate, flunisolide, flucortolone,fluticasone propionate, hydrocortisone, methylprednisolone,prednisolone, prednisone, triamcinolone, acetazolamide, amiloride,bendrofluazide, bumetanide, chlorothiazide, chlorthalidone, ethacrynicacid, frusemide, metolazone, spironolactone, triamterene, bromocriptinemesylate, lysuride maleate, bisacodyl, cimetidine, cisapride,diphenoxylate HCl, domperidone, famotidine, loperamide, mesalazine,nizatidine, omeprazole, ondansetron HCL, ranitidine HCl, sulphasalazine,acrivastine, astemizole, cinnarizine, cyclizine, cyproheptadie HCl,dimenhydrinate, flunarizine HCl, loratadine, meclozine HCl, oxatomide,terfenadine, bezafibrate, clofibrate, fenofibrate, gemfibrozil,probucol, amyl nitrate, glyceryl trinitrate, isosorbide dinitrate,isosorbide mononitrate, pentaerythritol tetranitrate, betacarotene,vitamin A, vitamin B 2, vitamin D, vitamin E, vitamin K, codeine,dextropropyoxyphene, diamorphine, dihydrocodeine, meptazinol, methadone,morphine, nalbuphine, pentazocine, clomiphene citrate, danazol, ethinylestradiol, medroxyprogesterone acetate, mestranol, methyltestosterone,norethisterone, norgestrel, estradiol, conjugated oestrogens,progesterone, stanozolol, stibestrol, testosterone, tibolone,amphetamine, dexamphetamine, dexfenfluramine, fenfluramine, andmazindol.

Uses and Pharmaceutical Compositions

The particles described herein may be employed in any suitableapplication. In some cases, the particles are part of pharmaceuticalcompositions (e.g., as described herein), for example, those used todeliver a pharmaceutical agent (e.g., a drug, therapeutic agent,diagnostic agent, imaging agent) through or to mucus or a mucosalsurface. A pharmaceutical composition may comprise at least one particledescribed herein and one or more pharmaceutically acceptable excipientsor carriers. The composition may be used in treating, preventing, and/ordiagnosing a condition in a subject, wherein the method comprisesadministering to a subject the pharmaceutical composition. A subject orpatient to be treated by the articles and methods described herein maymean either a human or non-human animal, such as primates, mammals, andvertebrates.

Methods involving treating a subject may include preventing a disease,disorder or condition from occurring in the subject which may bepredisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it; inhibiting the disease, disorder orcondition, e.g., impeding its progress; and relieving the disease,disorder, or condition, e.g., causing regression of the disease,disorder and/or condition. Treating the disease or condition includesameliorating at least one symptom of the particular disease orcondition, even if the underlying pathophysiology is not affected (e.g.,such treating the pain of a subject by administration of an analgesicagent even though such agent does not treat the cause of the pain).

In some embodiments, a pharmaceutical composition described herein isdelivered to a mucosal surface in a subject and may pass through amucosal barrier in the subject (e.g., mucus), and/or may exhibitprolonged retention and/or increased uniform distribution of theparticles at mucosal surfaces, e.g., due to reduced mucoadhesion.Non-limiting examples of mucosal tissues include oral (e.g., includingthe buccal and esophageal membranes and tonsil surface), ophthalmic,gastrointestinal (e.g., including stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., including nasal,pharyngeal, tracheal and bronchial membranes), genital (e.g., includingvaginal, cervical and urethral membranes).

Pharmaceutical compositions described herein and for use in accordancewith the articles and methods described herein may include apharmaceutically acceptable excipient or carrier. A pharmaceuticallyacceptable excipient or pharmaceutically acceptable carrier may includea non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any suitable type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose, and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose, andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; detergents such as Tween 80; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator. As would be appreciated by one of skill in this art, theexcipients may be chosen based on the route of administration asdescribed below, the pharmaceutical agent being delivered, time courseof delivery of the agent, etc.

Pharmaceutical compositions containing the particles described hereinmay be administered to a subject via any route known in the art. Theseinclude, but are not limited to, oral, sublingual, nasal, intradermal,subcutaneous, intramuscular, rectal, vaginal, intravenous,intraarterial, intracisternally, intraperitoneal, intravitreal,periocular, topical (as by powders, creams, ointments, or drops), bucaland inhalational administration. In some embodiments, compositionsdescribed herein may be administered parenterally as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. As would be appreciated by one of skillin this art, the route of administration and the effective dosage toachieve the desired biological effect may be determined by the agentbeing administered, the target organ, the preparation beingadministered, time course of administration, disease being treated,intended use, etc.

As an example, the particles may be included in a pharmaceuticalcomposition to be formulated as a nasal spray, such that thepharmaceutical composition is delivered across a nasal mucus layer. Asanother example, the particles may be included in a pharmaceuticalcomposition to be formulated as an inhaler, such that the pharmaceuticalcompositions is delivered across a pulmonary mucus layer. As anotherexample, if compositions are to be administered orally, it may beformulated as tablets, capsules, granules, powders, or syrups.Similarly, the particles may be included in a pharmaceutical compositionthat is to be delivered via ophthalmic, gastrointestinal, nasal,respiratory, rectal, urethral and/or vaginal tissues.

For application by the ophthalmic mucous membrane route, subjectcompositions may be formulated as eye drops or eye ointments. Theseformulations may be prepared by conventional means, and, if desired, thesubject compositions may be mixed with any conventional additive, suchas buffering or pH-adjusting agents, tonicity adjusting agents,viscosity modifiers, suspension stabilizers, preservatives, and otherpharmaceutical excipients. In addition, in certain embodiments, subjectcompositions described herein may be lyophilized or subjected to anotherappropriate drying technique such as spray drying.

In some embodiments, particles described herein that may be administeredin inhalant or aerosol formulations comprise one or more pharmaceuticalagents, such as adjuvants, diagnostic agents, imaging agents, ortherapeutic agents useful in inhalation therapy. The particle size ofthe particulate medicament should be such as to permit inhalation ofsubstantially all of the medicament into the lungs upon administrationof the aerosol formulation and may be, for example, less than about 20microns, e.g., in the range of about 1 to about 10 microns, e.g., about1 to about 5 microns, although other ranges are also possible. Theparticle size of the medicament may be reduced by conventional means,for example by milling or micronisation. Alternatively, the particulatemedicament can be administered to the lungs via nebulization of asuspension. The final aerosol formulation may contain, for example,between 0.005-90% w/w, between 0.005-50%, between 0.005-10%, betweenabout 0.005-5% w/w, or between 0.01-1.0% w/w, of medicament relative tothe total weight of the formulation. Other ranges are also possible.

It is desirable, but by no means required, that the formulationsdescribed herein contain no components which may provoke the degradationof stratospheric ozone. In particular, in some embodiments, propellantsare selected that do not contain or do not consist essentially ofchlorofluorocarbons such as CCl₃F, CCl₂F₂, and CF₃CCl₃.

The aerosol may comprise propellant. The propellant may optionallycontain an adjuvant having a higher polarity and/or a higher boilingpoint than the propellant. Polar adjuvants which may be used include(e.g., C₂₋₆) aliphatic alcohols and polyols such as ethanol,isopropanol, and propylene glycol, preferably ethanol. In general, onlysmall quantities of polar adjuvants (e.g., 0.05-3.0% w/w) may berequired to improve the stability of the dispersion—the use ofquantities in excess of 5% w/w may tend to dissolve the medicament.Formulations in accordance with the embodiments described herein maycontain less than 1% w/w, e.g., about 0.1% w/w, of polar adjuvant.However, the formulations described herein may be substantially free ofpolar adjuvants, especially ethanol. Suitable volatile adjuvants includesaturated hydrocarbons such as propane, n-butane, isobutane, pentane andisopentane and alkyl ethers such as dimethyl ether. In general, up to50% w/w of the propellant may comprise a volatile adjuvant, for example1 to 30% w/w of a volatile saturated C₁-C₆ hydrocarbon. Optionally, theaerosol formulations according to the invention may further comprise oneor more surfactants. The surfactants can be physiologically acceptableupon administration by inhalation. Within this category are includedsurfactants such as L-α-phosphatidylcholine (PC),1,2-dipalmitoylphosphatidycholine (DPPC), oleic acid, sorbitantrioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylenesorbitan monolaurate, polyoxyethylene sorbitan monooleate, naturallecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether,lauryl polyoxyethylene ether, block copolymers of oxyethylene andoxypropylene, synthetic lecithin, diethylene glycol dioleate,tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glycerylmonooleate, glyceryl monostearate, glyceryl monoricinoleate, cetylalcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridiniumchloride, benzalkonium chloride, olive oil, glyceryl monolaurate, cornoil, cotton seed oil, and sunflower seed oil.

The formulations described herein may be prepared by dispersal of theparticles in the selected propellant and/or co-propellant in anappropriate container, e.g., with the aid of sonication. The particlesmay be suspended in co-propellant and filled into a suitable container.The valve of the container is then sealed into place and the propellantintroduced by pressure filling through the valve in the conventionalmanner. The particles may be thus suspended or dissolved in a liquefiedpropellant, sealed in a container with a metering valve and fitted intoan actuator. Such metered dose inhalers are well known in the art. Themetering valve may meter 10 to 500 μL and preferably 25 to 150 μL. Incertain embodiments, dispersal may be achieved using dry powder inhalers(e.g., spinhaler) for the particles (which remain as dry powders). Inother embodiments, nanospheres, may be suspended in an aqueous fluid andnebulized into fine droplets to be aerosolized into the lungs.

Sonic nebulizers may be used because they minimize exposing the agent toshear, which may result in degradation of the particles. Ordinarily, anaqueous aerosol is made by formulating an aqueous solution or suspensionof the particles together with conventional pharmaceutically acceptablecarriers and stabilizers/surface-altering agents. The carriers andstabilizers/surface-altering agents vary with the requirements of theparticular composition, but typically include non-ionic surfactants(Tweens, Pluronic®, or polyethylene glycol), innocuous proteins likeserum albumin, sorbitan esters, oleic acid, lecithin, amino acids suchas glycine, buffers, salts, sugars, or sugar alcohols. Aerosolsgenerally are prepared from isotonic solutions.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredients (i.e.,microparticles, nanoparticles, liposomes, micelles, polynucleotide/lipidcomplexes), the liquid dosage forms may contain inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Incertain embodiments, the particles are suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween80.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration can be suppositorieswhich can be prepared by mixing the particles with suitablenon-irritating excipients or carriers such as cocoa butter, polyethyleneglycol, or a suppository wax which are solid at ambient temperature butliquid at body temperature and therefore melt in the rectum or vaginalcavity and release the particles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theparticles described herein, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles describedherein, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

The particles described herein comprising a pharmaceutical agent may beadministered to a subject to be delivered in an amount sufficient todeliver to a subject a therapeutically effective amount of anincorporated pharmaceutical agent as part of a diagnostic, prophylactic,or therapeutic treatment. In general, an effective amount of apharmaceutical agent or component refers to the amount necessary toelicit the desired biological response. The desired concentration ofpharmaceutical agent in the particle will depend on numerous factors,including, but not limited to, absorption, inactivation, and excretionrates of the drug as well as the delivery rate of the compound from thesubject compositions, the desired biological endpoint, the agent to bedelivered, the target tissue, etc. It is to be noted that dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions. Typically, dosingwill be determined using techniques known to one skilled in the art.

The concentration and/or amount of any pharmaceutical agent to beadministered to a subject may be readily determined by one of ordinaryskill in the art. Known methods are also available to assay local tissueconcentrations, diffusion rates from particles and local blood flowbefore and after administration of the therapeutic formulation.

The compositions and/or formulations described herein may have anysuitable osmolarity. In some embodiments, a composition and/orformulation described herein may have an osmolarity of at least about 0mOsm/L, at least about 5 mOsm/L, at least about 25 mOsm/L, at leastabout 50 mOsm/L, at least about 75 mOsm/L, at least about 100 mOsm/L, atleast about 150 mOsm/L, at least about 200 mOsm/L, at least about 250mOsm/L, or at least about 310 mOsm/L. In certain embodiments, acomposition and/or formulation described herein may have an osmolarityof less than or equal to about 310 mOsm/L, less than or equal to about250 mOsm/L, less than or equal to about 200 mOsm/L, less than or equalto about 150 mOsm/L, less than or equal to about 100 mOsm/L, less thanor equal to about 75 mOsm/L, less than or equal to about 50 mOsm/L, lessthan or equal to about 25 mOsm/L, or less than or equal to about 5mOsm/L. Combinations of the above-referenced ranges are also possible(e.g., an osmolarity of at least about 0 mOsm/L and less than or equalto about 50 mOsm/L). Other ranges are also possible. The osmolarity ofthe composition and/or formulation can be varied by changing, forexample, the concentration of salts present in the solvent of thecomposition and/or formulation.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1

The following describes a non-limiting example of a method of formingnon-polymeric solid particles into mucus-penetrating particles. Pyrene,a hydrophobic naturally fluorescent compound, was used as the coreparticle and was prepared by a nanomilling process in the presence ofvarious stabilizers. The stabilizers acted as surface-altering agentsand formed coatings around the core particles. Differentstabilizers/surface-altering agents were evaluated to determineeffectiveness of the coated particles in penetrating mucus.

Pyrene was nanomilled in aqueous dispersions in the presence of variousstabilizers/surface-altering agents to determine whether certainstabilizers/surface-altering agents can: 1) aid particle size reductionto several hundreds of nanometers and 2) physically (non-covalently)coat the surface of generated nanoparticles with a mucoinert coatingthat would minimize particle interactions with mucus constituents andprevent mucus adhesion. In these experiments, thestabilizers/surface-altering agents acted as a coating around the coreparticles, and the resulting particles were tested for their mobility inmucus, although in other embodiments, the stabilizers/surface-alteringagents may be exchanged with other surface-altering agents that canincrease mobility of the particles in mucus. Thestabilizers/surface-altering agents tested included a variety ofpolymers, oligomers, and small molecules listed in

Table 2, including pharmaceutically relevant excipients such aspoly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) blockcopolymers (Pluronics®), polyvinylpyrrolidones (Kollidon), andhydroxypropyl methylcellulose (Methocel), etc.

TABLE 2 Stabilizers/surface-altering agents tested with Pyrene as amodel compound. Polymeric Stabilizers/surface-altering agents Acronym orGrade or Molecular Stabilizer Trade Name Weight Chemical StructurePoly(ethylene oxide)- poly(propylene oxide)- poly(ethylene oxide) blockcopolymers Pluronic ® F127, F108, F68, F87, F28, P123, P105, P103, P65,L121, L101, L81, L44, L31

Polyvinylpyrrolidone PVP Kollidon 17 (9K), Kollidon 25 (26K), Kollindon30 (43K)

PVA-poly(ethylene glycol) graft-copolymer Kollicoat IR

Hydropropyl methylcellulose HPMC Methocel E50, Methocel K100

Oligomeric Stabilizers/surface-altering agents Tween 20

Tween 80

Solutol HS 15

Triton X100

Tyloxapol

Cremophor RH 40 Small Molecule Stabilizers/surface-altering argents Span20

Span 80

Octyl glucoside

Cetytrimethylammonium bromide (CTAB)

Sodium Dodecyl sulfate (SDS)

An aqueous dispersion containing pyrene and one of thestabilizers/surface-altering agents listed above was milled with millingmedia until particle size was reduced below 500 nm. Table 3 listsparticle size characteristics of pyrene particles obtained bynanomilling in the presence of the various stabilizers/surface-alteringagents. Particle size was measured by dynamic light scattering. WhenPluronics® L101, L81, L44, L31, Span 20, Span 80, or Octyl glucosidewere used as stabilizers/surface-altering agents, stable nanosuspensionscould not be obtained. Therefore, these stabilizers/surface-alteringagents were excluded from further investigation due to their inabilityto effectively aid particle size reduction.

TABLE 3 Particle size measured by DLS in nanosuspensions obtained bymilling of Pyrene with various stabilizers/surface-altering agents.Stabilizer N-Ave. D (nm) Pluronic ® F127 239 Pluronic ® F108 267Pluronic ® P105 303 Pluronic ® P103 319 Pluronic ® P123 348 Pluronic ®L121 418 Pluronic ® F68 353 Pluronic ® P65 329 Pluronic ® F87 342Pluronic ® F38 298 Pluronic ® L101 not measurable* Pluronic ® L81 notmeasurable* Pluronic ® L44 not measurable* Pluronic ® L31 notmeasurable* PVA 13K 314 PVA 31K 220 PVA 85K 236 Kollicoat IR 192Kollidon 17 (PVP 9K) 163 Kollidon 25 (PVP 26K) 210 Kollindon 30 (PVP43K) 185 Methocel E50 160 Methocel K100 216 Tween 20 381 Tween 80 322Solutol HS 378 Triton X100 305 Tyloxapol 234 Cremophor RH40 373 SDS 377CTAB 354 Span 20 not measurable* Span 80 not measurable* Octyl glucosidenot measurable* *milling with Pluronics ® L101, L81, L44, L31, Span 20,Span 80, Octyl glucoside failed to effectively reduce pyrene particlesize and produce stable nanosuspensions.

The mobility and distribution of pyrene nanoparticles from the producednanosuspensions in human cervicovaginal mucus (CVM) were characterizedusing fluorescence microscopy and multiple particle tracking software.In a typical experiment, ≤0.5 uL of a nanosuspension (diluted ifnecessary to the surfactant concentration of ˜1%) was added to 20 μl offresh CVM along with controls. Conventional nanoparticles (200 nmyellow-green fluorescent carboxylate-modified polystyrene microspheresfrom Invitrogen) were used as a negative control to confirm the barrierproperties of the CVM samples. Red fluorescent polystyrene nanoparticlescovalently coated with PEG 5 kDa were used as a positive control withwell-established MPP behavior. Using a fluorescent microscope equippedwith a CCD camera, 15 s movies were captured at a temporal resolution of66.7 ms (15 frames/s) under 100× magnification from several areas withineach sample for each type of particles: sample (pyrene), negativecontrol, and positive control (natural blue fluorescence of pyreneallowed observing of pyrene nanoparticles separately from the controls).Next, using an advanced image processing software, individualtrajectories of multiple particles were measured over a time-scale of atleast 3.335 s (50 frames). Resulting transport data are presented herein the form of trajectory-mean velocity V_(mean), i.e., velocity of anindividual particle averaged over its trajectory, and ensemble-averagevelocity <V_(mean)>, i.e., V_(mean) averaged over an ensemble ofparticles. To enable easy comparison between different samples andnormalize velocity data with respect to natural variability inpenetrability of CVM samples, relative sample velocity <V_(mean)>_(rel),was determined according to the formula shown in Equation 1.

Prior to quantifying mobility of the produced pyrene nanoparticles,their spatial distribution in the mucus sample was assessed bymicroscopy at low magnifications (10×, 40×). It was found thatpyrene/Methocel nanosuspensions did not achieve uniform distribution inCVM and strongly aggregated into domains much larger than the mucus meshsize (data not shown). Such aggregation is indicative of mucoadhesivebehavior and effectively prevents mucus penetration. Therefore, furtherquantitative analysis of particle mobility was deemed unnecessary.Similarly to the positive control, all other tested pyrene/stabilizersystems achieved a fairly uniform distribution in CVM. Multiple particletracking confirmed that in all tested samples the negative controls werehighly constrained, while the positive controls were highly mobile asdemonstrated by <V_(mean)> for the positive controls being significantlygreater than those for the negative controls (Table 4).

TABLE 4 Ensemble-average velocity <V_(mean)> (um/s) and relative samplevelocity <V_(mean)>_(rel) for pyrene/stabilizer nanoparticles (sample)and controls in CVM. Negative Positive Sample Control Control Sample(relative) Stabilizer <V_(mean)> SD <V_(mean)> SD <V_(mean)> SD<V_(mean)>_(rel) SD Pluronic F127 0.58 0.18 5.97 0.54 6.25 0.72 1.050.18 Pluronic F108 0.43 0.64 5.04 1.88 4.99 1.47 0.99 0.55 Pluronic P1050.56 0.52 4.38 1.36 4.47 2.11 1.02 0.69 Pluronic P103 0.58 0.77 4.5 2.014.24 1.95 0.93 0.74 Pluronic P123 0.56 0.44 4.56 1.44 3.99 1.66 0.860.54 Pluronic L121 0.42 0.3 4.27 2.04 0.81 0.51 0.10 0.16 Pluronic F680.56 0.52 4.38 1.36 0.81 0.7 0.07 0.23 Pluronic P65 0.26 0.25 4.52 2.150.53 0.56 0.06 0.15 Pluronic F87 0.95 1.6 5.06 1.34 0.74 0.78 −0.05−0.43 Pluronic F38 0.26 0.1 5.73 0.84 0.54 0.29 0.05 0.06 Pluronic L101*Pluronic L81* Pluronic L44* Pluronic L31* Kollicoat IR 0.62 0.62 5.390.55 0.92 0.81 0.06 0.22 Kollidon 17 1.69 1.8 5.43 0.98 0.82 0.59 −0.23−0.52 Kollidon 25 0.41 0.34 5.04 0.64 1.29 1.09 0.19 0.25 Kollindon 300.4 0.2 4.28 0.57 0.35 0.11 −0.01 0.06 Methocel E50** Methocel K100**Tween 20 0.77 0.93 5.35 1.76 1.58 2.02 0.18 0.49 Tween 80 0.46 0.34 3.351.89 0.94 0.5 0.17 0.24 Solutol HS 0.42 0.13 3.49 0.5 0.8 0.6 0.12 0.20Triton X100 0.26 0.13 4.06 1.11 0.61 0.19 0.09 0.07 Tyloxapol 0.5 0.53.94 0.58 0.42 0.23 −0.02 −0.16 Cremophor RH40 0.48 0.21 3.2 0.97 0.490.24 0.00 0.12 Span 20* Span 80* Octyl glucoside* SDS 0.3 0.12 5.99 0.840.34 0.15 0.01 0.03 CTAB 0.39 0.09 4.75 1.79 0.32 0.31 −0.02 −0.07 *Didnot produce stable nanosuspensions, hence not mucus-penetrating(velocity in CVM not measured) **Aggregated in CVM, hence notmucus-penetrating (velocity in CVM not measured)

It was discovered that nanoparticles obtained in the presence of certain(but, importantly, not all) stabilizers/surface-altering agents migratethrough CVM at the same rate or nearly the same velocity as the positivecontrol. Specifically, pyrene nanoparticles stabilized with Pluronics®F127, F108, P123, P105, and P103 exhibited <V_(mean)> that exceededthose of the negative controls by approximately an order of magnitudeand were indistinguishable, within experimental error, from those of thepositive controls, as shown in Table 4 and FIG. 2A. For these samples,<V_(mean)>_(rel) values exceeded 0.5, as shown in FIG. 2B.

On the other hand, pyrene nanoparticles obtained with the otherstabilizers/surface-altering agents were predominantly or completelyimmobilized as demonstrated by respective <V_(mean)>_(rel) values of nogreater than 0.4 and, with most stabilizers/surface-altering agents, nogreater than 0.1 (Table 4 and FIG. 2B). Additionally, FIGS. 3A-3D arehistograms showing distribution of V_(mean) within an ensemble ofparticles. These histograms illustrate muco-diffusive behavior ofsamples stabilized with Pluronic® F127 and Pluronic® F108 (similarhistograms were obtained for samples stabilized with Pluronic® P123,P105, and P103, but are not shown here) as opposed to muco-adhesivebehavior of samples stabilized with Pluronic® 87, and Kollidon 25(chosen as representative muco-adhesive samples).

To identify the characteristics of Pluronics® that render pyrenenanocrystals mucus penetrating, <V_(mean)>_(rel) of the Pyrene/Pluronic®nanocrystals was mapped with respect to molecular weight of the PPOblock and the PEO weight content (%) of the Pluronics® used (FIG. 4). Itwas concluded that at least those Pluronics® that have the PPO block ofat least 3 kDa and the PEO content of at least about 30 wt % renderedthe nanocrystals mucus-penetrating. Without wishing to be bound by anytheory, it is believed that the hydrophobic PPO block can provideeffective association with the surface of the core particles if themolecular weight of that block is sufficient (e.g., at least about 3 kDain some embodiments); while the hydrophilic PEO blocks are present atthe surface of the coated particles and can shield the coated particlesfrom adhesive interactions with mucin fibers if the PEO content of thePluronic® is sufficient (e.g., at least 30 wt % in some embodiments). Asdescribed herein, in some embodiments the PEO content of thesurface-altering agent may be chosen to be greater than about 10 wt %(e.g., at least about 15 wt %, or at least about 20 wt %), as a 10 wt %PEO portion rendered the particles mucoadhesive.

Example 2

This example describes the formation of mucus-penetrating particlesusing various non-polymeric solid particles.

The technique described in Example 1 was applied to other non-polymericsolid particles to show the versatility of the approach. F127 was usedas the surface-altering agent for coating a variety of activepharmaceuticals used as core particles. Sodium dodecyl sulfate (SDS) waschosen as a negative control so that each drug was compared to asimilarly sized nanoparticle of the same compound. An aqueous dispersioncontaining the pharmaceutical agent and Pluronic® F127 or SDS was milledwith milling media until particle size was reduced below 300 nm. Table 5lists the particle sizes for a representative selection of drugs thatwere milled using this method.

TABLE 5 Particle sizes for a representative selection of drugs milled inthe presence of SDS and F127. Drug Stabilizer Z-Ave D (nm) PDIFluticasone F127 203 0.114 Propionate SDS 202 0.193 Furosemide F127 2170.119 SDS 200 0.146 Itraconazole F127 155 0.158 SDS 168 0.163Prednisolone F127 273 0.090 SDS 245 0.120 Loteprednol F127 241 0.123Etabonate SDS 241 0.130 Budesonide F127 173 0.112 SDS 194 0.135Indomethacin F127 225 0.123 SDS 216 0.154

In order to measure the ability of drug nanoparticles to penetrate mucusa new assay was developed which measures the mass transport ofnanoparticles into a mucus sample. Most drugs are not naturallyfluorescent and are therefore difficult to measure with particletracking microscopy techniques. The newly-developed bulk transport assaydoes not require the analyzed particles to be fluorescent or labeledwith dye. In this method, 20 μL of CVM is collected in a capillary tubeand one end is sealed with clay. The open end of the capillary tube isthen submerged in 20 μL of an aqueous suspension of particles which is0.5% w/v drug. After the desired time, typically 18 hours, the capillarytube is removed from the suspension and the outside is wiped clean. Thecapillary containing the mucus sample is placed in an ultracentrifugetube. Extraction media is added to the tube and incubated for 1 hourwhile mixing which removes the mucus from the capillary tube andextracts the drug from the mucus. The sample is then spun to removemucins and other non-soluble components. The amount of drug in theextracted sample can then be quantified using HPLC. The results of theseexperiments are in good agreement with those of the microscopy method,showing clear differentiation in transport between mucus penetratingparticles and conventional particles (CP). The transport results for arepresentative selection of drugs are shown in FIG. 5. These resultscorroborate microscopy/particle tracking findings with Pyrene anddemonstrate the extension to common active pharmaceutical compounds;coating non-polymeric solid nanoparticles with F127 enhances mucuspenetration.

In Examples 1-2, cervicovaginal mucus (CVM) samples were obtained fromhealthy females volunteers age 18 years or older. CVM was collected byinserting a Softcup® menstrual collection cup into the vaginal tract asdescribed by the product literature for between 30 seconds and 2minutes. After removal the CVM was then collected from the Softcup® bygentle centrifugation at ˜30 xG to ˜120 xG in a 50 mL centrifuge tube.In Example 1, CVM was used undiluted and fresh (stored for no longerthan 7 days under refrigerated conditions). Barrier and transport of allCVM samples used in Example 1 were verified with negative (200 nmcarboxylated polystyrene particles) and positive (200 nm polystyreneparticles modified with PEG 5K) controls. In Example 2, CVM waslyophilized and reconstituted. In Example 2, mucus was frozen at −50° C.and then lyophilized to dryness. Samples were then stored at −50° C.Before use, the mucus was reconstituted by grinding the solid into afine powder using a mortar and pestle followed by water addition to afinal volume equal to the original volume to 2 times the originalvolume. The reconstituted mucus was then incubated at 4° C. for 12 hoursand used as described in Example 2. Barrier and transport of all CVMsamples used in Example 2 were verified with negative (200 nmcarboxylated polystyrene particles) and positive (F127 coated 200 nmpolystyrene particles) controls.

Example 3

This example describes the formation of mucus-penetrating particlesusing a core comprising the drug loteprednol etabonate (LE).

In order to demonstrate the value of enhanced mucus penetration in thedelivery of non-polymeric solid particles, an MPP formulation ofloteprednol etabonate (LE MPP; LE particles coated with Pluronic® F127made by the method described in Example 2) was compared to the currentlymarketed formulation, Lotemax®. Lotemax® is a steroid eye drop approvedfor the treatment of surface ocular inflammation. Conventionalparticles, such as those in Lotemax®, are extensively trapped by theperipheral rapidly-cleared mucus layer in the eye and, hence, are alsorapidly cleared. LE MPP are able to avoid adhesion to, and effectivelypenetrate through, mucus to facilitate sustained drug release directlyto underlying tissues. Enhancing drug exposure at the target site wouldallow the overall dose to be reduced, increasing patient compliance andsafety. In vivo, a single topical instillation of LE MPP to New Zealandwhite rabbits produced significantly higher drug levels in palpebralconjunctiva, bulbar conjunctiva, and cornea compared to an equivalentdose of Lotemax® (FIGS. 6A-6C). At 2 hours LE levels from MPP are 6, 3,and 8 times higher than from Lotemax® (palpebral, bulbar, and cornea,respectively). Notably, LE levels from MPP are approximately 2 timeshigher at 2 hours than levels from Lotemax® at 30 minutes. These resultsdemonstrate the utility of the non-polymeric solid MPP approach.

Example 4

This example describes the formation of mucus-penetrating particlesincluding a core comprising curcumin (CUR).

Molecules with various solubilities were selected as model therapeuticagents for forming particles having a core of a solid pharmaceuticalagent. One of them, curcumin, is suggested to have antioxidant,antitumor and anti-inflammatory properties. It is an interestingcandidate not only because of its broad potential medical applications,but also its high hydrophobicity and natural fluorescence. The formerfeature means that CUR is poorly soluble in aqueous solutions, while thelatter allows rapid and label-free detection and characterization of theparticles. The particles were coated with surfactants (e.g., Pluronic®F127, abbreviated as F127 in Examples 4 and 5) to render themmucus-penetrating.

A simple procedure based on ultrasonication was developed to formulateparticles of CUR. Briefly, 5 mg CUR was dispersed in 2 mL aqueoussolution containing F127 (or other surfactants) in a 7 mL scintillationvial. The suspension was sonicated in a water bath for 20 min. Thecurcumin suspension was then sonicated using an ultra-sonicator with a 3mm stepped probe at 100% amplitude for 30 min. The suspension wascentrifuged at 2000 rpm for 10 min to remove unbroken crystals. Thesupernatant was stored at 4 C for 2 hours. The supernatant wascentrifuged at 16,500 rpm for 20 min, and then the pellet was collected.It was noticed that without sufficient incubation time before particlecollection, the diffusivity of the coated particles was much lower (datanot shown), suggesting the importance of a dense F127 coating in thegeneration of mucus-penetrating particles.

Table 6 and (FIGS. 7A-7B) summarize the physicochemical properties ofthe resulting coated CUR particles prepared using the above method. CURparticles formulated in 1% (w/v) F127 (CUR-1% F127 particles) possessedan average size of 133 nm, which was consistent with the observationthrough TEM images (FIG. 7B). The Zeta-potential was close to neutral.As F127 concentration in the CUR suspension during ultra-sonication wasdecreased, CUR particle size and polydispersity (PDI) each increased,likely as a result of reduced F127 coating density leading to weakenedstabilizing effect on CUR particles (Table 6). There was little effectof F127 concentration on particle Zeta-potential, which is likelyattributed to the deionization of curcumin at pH 4. Powder-XRDmeasurement on CUR-1% F127 particles indicated that the chemicalstructure and crystallinity of CUR were not altered by eitherultrasonication or the incorporation of F127 (FIG. 7A).

TABLE 6 Size and Zeta-potential of CUR particles prepared in differentconcentrations of F127 and their diffusivity in human cervicovaginalmucus (D_(m)) compared to in water (D_(w)) Sample Size PolydispersityZeta-potential Description*** (nm)* Index (mV)* D_(w)/D_(m)** CUR-1%F127 133 ± 12 0.33 ± 0.05 −0.8 ± 0.1 9 particles CUR-0.1% 154 ± 4 0.50 ±0.02 −1.4 ± 0.4 11 F127 particles CUR-0.01% 176 ± 11 0.57 ± 0.03 −1.6 ±0.3 35 F127 particles CUR-0.001% 184 ± 30 0.72 ± 0.03 −1.3 ± 0.5 10000F127 particles *Size and Zeta-potential were measured in 10 mM NaCl (pH= 4) via dynamic light scattering and laser Doppler anemometry,respectively. Data represent mean ± standard error (n = 3). **D_(w) iscalculated from the Stokes-Einstein equation using average particle sizeand D_(m) is the geometric ensemble average effective diffusivity(<Deff>) calculated at a time scale of 1 s. ***The percentage of F127indicates its concentration (% w/v) during the preparation of CURparticles.

To examine the mucus-penetrating properties of CUR-F127 particles, thetransport of CUR-1% F127 particles was studied using multiple particletracking (MPT) in both human cervicovaginal mucus (CVM) and human cysticfibrosis sputum (CFS) samples. In brief, the particles were added tomucus samples and their motion was recorded using high resolutionepifluorescence microscopy. Their trajectories and transport rates werethen analyzed and quantified. FIGS. 8A-8B show the time-dependentensemble averaged geometric mean square displacement (<MSD>) of CUR-1%F127 particles in both CVM and CFS. 200 nm PEGylated (PEG) andcarboxylated (COOH) polystyrene (PS) particles were selected asmuco-inert and muco-adhesive controls, respectively. For the entirerange of studied time scales, the <MSD> of CUR-1% F127 particles wascomparable to that of PSPEG in CVM, and significantly higher than thatof PSCOOH in both types of mucus samples. At a time scale of is, the<MSD> of CUR-1% F127 particles was 4400-fold and 220-fold higher thanthat of PSCOOH in CVM and CFS, respectively. The geometric ensembleaveraged effective diffusivity (<Deff>) of CUR-1% F127 particles at atime scale of is was only 9-fold lower than the calculated theoreticaldiffusivity in water (Table 6).

To further investigate the effect of the coating density of F127 on thetransport of CUR particles in mucus, the particles were formulated atvarious F127 concentrations and their diffusivities were characterizedin human CVM (FIG. 9, Table 6). The <Deff> of CUR-0.1% and 0.01% F127particles were either similar or slightly reduced as compared to that ofCUR-1% F127 particles, but the transport of CUR-0.001% F127 particleswas dramatically hindered (FIG. 9). The diffusivity of CUR-0.001% F127particles was 10000-fold slower in human CVM as compared to in water,and about 1000-fold lower than that of CUR-1% F127 particles in CVM. Ingeneral, reducing the F127 concentration in the preparation of CURparticles resulted in a decrease of diffusivity, probably because thedecreased F127 concentration lowered the surface density of F127 (andthereby the PEG brushes) at equilibrium. It was not obvious that thiseffect would become so pronounced when the F127 concentration dippedbelow 0.01% (w/v).

TABLE 7 Size and Zeta-potential of CUR particles prepared in differentPluronics ® and their diffusivity in human cervicovaginal mucus (D_(m))compared to in water (D_(w)) Sample Description Size (nm)*Polydispersity Index Zeta-potential (mV)* D_(w)/D_(m)** CUR-1% F38particles 232 ± 46 0.56 ± 0.01 −1.2 ± 0.5 60 CUR-1% P65 particles 187 ±34 0.48 ± 0.05 −1.5 ± 0.3 1600 CUR-1% P68 particles 154 ± 20 0.53 ± 0.02−1.4 ± 0.5 150 CUR-1% P84 particles 138 ± 12 0.38 ± 0.02 −1.3 ± 0.5 30CUR-1% P85 particles 149 ± 16 0.43 ± 0.05 −1.3 ± 0.3 30 CUR-1% F88particles 130 ± 19 0.51 ± 0.04 −0.5 ± 0.5 20 CUR-1% F98 particles 123 ±34 0.50 ± 0.02 −0.8 ± 0.1 12 CUR-1% P103 particles 141 ± 4 0.50 ± 0.02−1.4 ± 0.3 8 CUR-1% P104 particles 110 ± 18 0.45 ± 0.01 −1.8 ± 0.6 12CUR-1% P105 particles  90 ± 8 0.54 ± 0.02 −1.0 ± 0.5 15 CUR-1% F108particles 135 ± 21 0.52 ± 0.03 −0.6 ± 0.1 11 CUR-1% P123 particles 123 ±11 0.60 ± 0.06 −2.3 ± 0.5 9 *Size and Zeta-potential of particles weremeasured in 10 mM NaCl (pH = 4) via dynamic light scattering and laserDoppler anemometry, respectively. Data represent mean ± standard error(n = 3). **D_(w) is calculated from the Stokes-Einstein equation usingaverage particle diameter and D_(m) is the ensemble average effectivediffusivity calculated at a time scale of 1 s.

A variety of Pluronics®, in addition to F127, have been used forpharmaceutical applications. Different Pluronics® were tested to see ifthey would work equally well, or if only certain ones would transformCUR particles into mucus-penetrating particles. Twelve additionalPluronics®, as listed (in order of increasing PPO MW) in Table 7, wereselected and the corresponding CUR particles were prepared. Theresulting size ranged from 90 to 232 nm, while mostly staying between100-150 nm. All the particle types showed a PDI larger than that ofCUR-1% F127 particles (0.33), falling mostly between 0.4-0.6. Theseresult implied that F127 may have the strongest stabilizing effect amongall the tested Pluronics®. The Zeta-potential was uniformly neutral forall the studied CUR particles.

The transport rates of the CUR particles coated with various Pluronics®were characterized in human CVM (Table 7). By comparing theirdiffusivity to that of CUR-1% F127 particles (Dw/Dm=9), they could beclustered into three groups: particle motion strongly hindered (F65,F68; Dw/Dm >100), particle motion hindered (F38, F84, F85, F88;20≤Dw/Dm≤100), and particles rapidly penetrating (F98, P103, P104, P105,F108, F123; Dw/Dm<20). The fact that the particles all possessed nearneutral surface charge, but that only formulations with certainPluronic® coatings exhibited strongly hindered diffusivity (e.g., F65and F68), suggests that the adhesiveness between these CUR particles andmucus components is likely dominated by hydrophobic interactions.

To identify the factors determining the diffusivity of CUR particlescoated with different Pluronics®, <Deff> (at a time scale of is) of theCUR particles was mapped with regards to the PPO and PEG MW of the usedPluronics® (FIG. 10A). A general increase of <Deff> was observed alongwith the growing length of PPO segment (Y-axis), yet no specific patternbetween <Deff> and the PEG MW (X-axis). Surprisingly, this transitionoccurred at a PPO MW around 2000 Da, which is smaller than previousfound for Pluronic® coated polystyrene particles. Particle diffusivityexhibited a strong correlation with the PPO MW (R=0.92), but littlecorrelation with the PEG MW (R=0.14). It is likely that Pluronics® withlonger PPO segments might have higher affinity to the hydrophobicsurface of CUR particles, thus anchor more tightly and provide denserand more stable shielding on the surface.

To explore the capability of surfactants other than Pluronics® topotentially generate mucus-penetrating particles, Tween 20, Tween 80 andVitamin E TPGS, all containing PEG segments, were tested.Characteristics of CUR particles prepared in these surfactants arelisted in Table 8. Although all three groups demonstrated particle sizearound 150 nm and Zeta-potential close to neutral, their diffusivity wasgreatly reduced in human CVM compared to in water. The lower transportrates might indicate ineffective coating of surfactant molecules on thesurface of CUR particles, which may be attributed to their shorterhydrophobic segments as compared to F127.

TABLE 8 Size and Zeta-potential of CUR particles prepared in surfactantsother than Pluronics ®, and their diffusivity in human cervicovaginalmucus (D_(m)) compared to in water (D_(w)) Diameter Polydispersity Zeta-Sample Description (nm)* Index potential (mV)* D_(w)/D_(m)** N_(b) ^(‡)N_(m) ^(‡) CUR-1% Tween20 153 0.56 −3.2 13000 1 1 particles CUR-1%Tween80 155 0.60 −2.9 480 1 1 particles CUR-1% VitE-TPGS^(†) 135 0.68−2.5 1700 1 1 particles *Size and Zeta-potential of particles weremeasured in 10 mM NaCl (pH = 4) via dynamic light scattering and laserDoppler anemometry, respectively. Data represent mean ± standard error(n = 3). **D_(w) is calculated from the Stokes-Einstein equation usingaverage particle size and D_(m) is the ensemble average effectivediffusivity calculated at a time scale of 1 s. ^(†)VitE-TPGS: Vitamin-ETPGS (Vitamin-ETPGS (d-alpha tocopheryl polyethylene glycol 1000succinate), from Antares Health Products, Inc. (Batavia, IL)) ^(‡)N_(b):number of batches of particles tested; N_(m): number of human CVMsamples tested.

In order to evaluate the ability of CUR particles to deliver CUR in asustained fashion, the release profile of CUR-1% F127 particles wascharacterized. In brief, a known amount of CUR particles was suspendedin phosphate buffered saline (PBS, pH 7.4) in a 50 mL tube with a layerof octanol added on top to extract the dissolved CUR. The suspension wasincubated at 37° C. with agitation. Octanol was collected and replacedat each time point. The concentration of CUR in octanol was determinedby fluorometry. As shown in FIG. 11, CUR-1% F127 particles providedcontinuous release for 24-48 hours in vitro. About 80% of the CURcontent was released within the first 24 hours.

Example 5

This example describes the development of mucus-penetrating particlesusing a hydrophobic drug, 5, 10, 15, 20-tetra(p-hydroxyphenyl)porphyrin(p-THPP).

In addition to CUR, the same method described in Example 4 was appliedthe hydrophobic drug, p-THPP. p-THPP is a therapeutic agent used forphotodynamic therapy to treat cancer and has been selected as a modelphotosensitizer in previous studies. Basic properties of p-THPP-1% F127particles, including size, Zeta-potential and diffusivity in human CVMwere determined following the procedures described before (Table 9).Similar to CUR-1% F127 particles, p-THPP-1% F127 particles exhibited asize of 187 nm and a close-to-neutral surface charge. The diffusivity ofp-THPP-1% F127 particles in human CVM was only 8-fold slower than thatin water, indicating that the particles are not immobilized by theadhesive components in mucus and are able to diffuse through the mucusgel with a comparable rate to that of CUR particles-1% F127.

TABLE 9 Size and Zeta-potential of p-THPP particles prepared inPluronic ® F127 and their diffusivity in human cervicovaginal mucus(D_(m)) compared to in water (D_(w)) Sample Size PolydispersityZeta-potential Description (nm)* Index (mV)* D_(w)/D_(m)** p-THPP-1%F127 187 ± 7 0.51 0.3 ± 0.4 8 particles *Size and Zeta-potential ofparticles were measured in 10 mM NaCl (pH = 4) via dynamic lightscattering and laser Doppler anemometry, respectively. Data representmean ± standard error (n = 3). **D_(w) is calculated from theStokes-Einstein equation using average particle size and D_(m) is theensemble average effective diffusivity calculated at a time scale of 1s.

Example 6

This example describes the development of mucus-penetrating particlesusing tenofovir (TFV) and acyclovir monophosphate (ACVp), highly watersoluble drugs.

Tenofovir (TFV) is a potent antiviral drug used to treat infectiousdiseases. Due to the fact that tenofovir (TFV) is highly water soluble,a method was developed for formulating mucus-penetrating particles oftenofovir. The water solubility of TFV is at least 15 mg/mL, soconventional techniques for preparation of insoluble particles or,alternatively, encapsulation into hydrophobic polymeric nanoparticles,were not successful. In order to decrease the water solubility of TFV,interactions between cations and nucleotides/nucleotide analogs wereexploited. TFV interacts very strongly with zinc cations (Zn), via thephosphonate group and the purine ring structure. This interaction withzinc causes TFV precipitation into crystals that can be stabilized withthe coatings described herein, halting aggregation and determining thecrystal surface properties. Additionally, Zn is naturally present invaginal fluid and has known antimicrobial properties recently extendedto include anti-HIV activity.

It was necessary to ensure that the TFV-Zn particles would exhibitextended release into buffer, because the crystal and coating are formedentirely of non-covalent interactions. When compared to a solution offree TFV, the particles exhibited much slower release (FIG. 12) from 100kDa dialysis membranes (about 40% over 24 h).

Next, TFV-Zn particles were formulated using either F127 or PVAcoatings. As seen in Table 10, the presence of the coatings stabilizedthe particles, as evidenced by the smaller average size and decreasedpolydispersity. The presence of the coatings on the surface is alsoimplied by the change in zeta potential toward a more neutral charge.Additionally, these particles were fluorescently labeled for imagingpurposes, via covalent attachment of an Alexa Fluor® dye to the freeamine on TFV. Crystals were found to be unstable when made at a ratio of1:50 labeled:unlabeled TFV, but were stable and visible via fluorescentmicroscopy at a ratio of 1:200 labeled:unlabeled TFV.

TABLE 10 Size and zeta potential of TFV particles Coating MPP/CP Size(nm) ζ potential (mV) 0.08% F127 MPP 154 ± 4 −8.7 ± 1.6 0.1% PVA CP 165± 14 −7.3 ± 0.4 None CP 328 ± 91 −21.5 ± 0.9 

After obtaining stable, fluorescently labeled particles, it wasdetermined whether the F127 coating would lead to improved particledistribution at a mucosal surface in animals, as was consistently seenwith coated polymeric nanoparticles. TFV particles coated with eitherF127 or PVA were administered into the vaginas of mice, then the micewere sacrificed, their vaginas dissected out and flattened on amicroscope slide, and imaged. As can be seen in FIGS. 13A-13B,F127-coated particles were well distributed over the entire vaginalsurface, whereas the PVA-coated particles show incomplete coverage ofthe vaginal surface, evident as a “striping” behavior indicative of notentering the vaginal folds (rugae).

TABLE 11 HSV-2 Vaginal Challenge Results Group Drug Form & Conc. Vehicle# Mice # Infected % Infected p-value** 1 Placebo PBS 100 88 88.0<0.000001 2 Soluble ACV (1 mg/mL) PBS 25 21 84.0 0.002 3 Soluble ACV (10mg/mL) PBS 100 62 62.0 0.10 4 Soluble ACV (10 mg/mL) Water 75 52 69.30.02 5 ACV-MPP (1 mg/mL)* Water 45 21 46.7 N/A *Size 65.3 ± 10 nm, ζpotential −6.3 ± 1.0 mV. **p-value vs. ACV-MPP.

Next MPP particles were produced using acyclovir monophosphate (ACVp)for use in testing the potential efficacy of MPP particles in preventinginfection with the Herpes Simplex Virus Type 2 (HSV-2). ACVp is notdependent on viral thymidine kinase for the initial phosphorylation stepthat can lead to viral resistance. This also gives ACVp anti-HIVactivity, although at low potency.

Female 6-8 week old CF-1 mice were subcutaneously injected withmedroxyprogesterone acetate, and one week later received 20 μL of testagent or PBS intravaginally with a fire-polished positive displacementcapillary pipette (Wiretrol, Drummond Scientific). Thirty minutes later,mice were challenged with 10 uL of inoculum containing HSV-2 strain G(ATCC #VR-734, 2.8×10⁷ TCID₅₀ per mL). HSV-2 was diluted 10-fold withBartel's medium to deliver 10 ID₅₀, a dose that typically infects ˜85%of control mice. Mice were assessed for infection three days later afterinoculation by culturing a PBS vaginal lavage on human foreskinfibroblasts (Diagnostic Hybrids, MRHF Lot #440318W), as describedpreviously (Cone R A et al., BMC Infect Dis 6:90, 2006). In this model,input (challenge) virus is no longer detectable in lavage fluid if it iscollected more than 12 hours after the challenge.

It was found that ACVp particles could be prepared in the presence ofzinc, via similar interactions as TFV (phosphate group and purine ring).Soluble ACVp or ACVp in the form of MPP nanocrystals was administered tomice 30 min prior to challenge with live HSV. Both the drug and thevirus were administered intravaginally. Soluble drug administered at thesame concentration as the MPP-drug was ineffective (84% infectedcompared to 88% of controls), whereas only 46.7% of mice in the MPP-druggroup were infected. Groups of mice given soluble drug at 10-times thedose given of MPP-drug were infected at a rate of 62% (drug in PBS) or69.3% (drug in water). Comparing soluble drug to MPP-drug administeredin the same vehicle (pure water), soluble drug was less protective(p=0.02) even at 10-times higher concentration than MPP-drug.

It is noted that stable nanocrystals of drugs with a free phosphategroup can be formed with Zn using both freeze-drying approaches andsonication procedures. For freeze drying, the drug was dissolved in anaqueous solution of F127. An amount of zinc acetate was added in therange of 1:50 to 1:5 (Zn: drug), and the solution immediately flashfrozen. The dried powder was reconstituted in water at the desiredconcentration. F127 concentrations both above (1%) and below (0.08%) thecritical micelle concentration (CMC) of F127 can be used to make stableTFV nanocrystals. However, the ACVp nanocrystals were much moresensitive to surfactant concentration. Stable nanocrystals could only beformed when using F127 concentrations below the CMC (˜0.1%). F127concentrations above the CMC caused significant aggregation andsedimentation of the ACVp nanocrystals, regardless of the formulationmethod tested.

Additionally, stable nanocrystals can be formed by adding excess zincacetate to drug solution. The precipitate is washed 3+ times bycentrifugation. The resultant slurry is sonicated using a probesonicator (without surfactant; bubbling causes aggregation andinstability). Other methods, such as milling, may also work. Surfactantis then added to stabilize the resultant nanocrystals. Without thepresence of surfactant, significant aggregation and sedimentationoccurs. Using this formulation technique, stable TFV nanocrystals couldbe made using F127 concentrations up to, for example, 1%. Similarly, inthese experiments, stable ACVp nanocrystals could only be formed usingF127 concentrations below the CMC (typically using 0.08%).

In Examples 4-6, undiluted cervicovaginal secretions were collected fromwomen with normal vaginal flora using a self-sampling menstrualcollection device following a protocol approved by the InstitutionalReview Board of the Johns Hopkins University. The device was insertedinto the vagina for ˜30 s, removed, and placed into a 50 mL centrifugetube. Samples were centrifuged at 1,000 rpm for 2 min to collect themucus secretions.

In Examples 4-6, particle transport rates were measured by analyzingtrajectories of fluorescent or fluorescently labeled particles, recordedusing an electron multiplying charge coupled device (EMCCD) camera(Evolve 512, Photometrics, Tucson, Ariz.) mounted on an invertedepifluorescence microscope (Zeiss, Thornwood, N.Y.) equipped with a 100×oil-immersion objective (N.A., 1.46) and the appropriate filters.Experiments were carried out in custom-made chamber slides, wherediluted particle solutions (0.0082% wt/vol) were added to 20 μL of freshmucus to a final concentration of 3% v/v (final particle concentration,8.25×10−7 wt/vol) and stablized at room temperature before microscopy.Trajectories of n≥100 particles were analyzed for each experiment, andat least three independent experiments were performed for eachcondition. Movies were captured with MetaMorph software (UniversalImaging, Glendale, Wis.) at a temporal resolution of 66.7 ms for 20 s.The tracking resolution was 10 nm, as determined by tracking thedisplacements of particles immobilized with a strong adhesive. Thecoordinates of nanoparticle centroids were transformed intotime-averaged MSD, calculated as <Δr2(τ)>=[x(t+τ)−x(t)]²+[y(t+τ)−y(t)]²,where x and y represent the nanoparticle coordinates at a given time andτ is the time scale or time lag. Distributions of MSDs and effectivediffusivities were calculated from this data. Particle penetration intoa mucus layer was modeled using Fick's second law and diffusioncoefficients obtained from tracking experiments.

Example 7

This example describes the development of mucus-penetrating particlesthat improve drug delivery to the mucosal surface of the mouse vagina.

Improved methods for sustained and more uniform drug delivery to thevagina may provide more effective prevention and treatment of conditionsthat impact women's health, such as cervical cancer, bacterialvaginosis, and sexually transmitted infections. For example, women aredisproportionately infected with HIV, partly owing to a lack offemale-controlled prevention methods. An easily administered, discreet,and effective method for protecting women against vaginal HIVtransmission could prevent millions of infections worldwide. However,vaginal folds, or “rugae”, that accommodate expansion during intercourseand child birth, are typically collapsed by intra-abdominal pressure,making the surfaces of these folds less accessible to drugs and drugcarriers. Poor distribution into the vaginal folds, even after simulatedintercourse, has been cited as a critical factor for failure to protectsusceptible vaginal surfaces from infection. Distribution over theentire susceptible target surface has been proven important forpreventing and treating infections. Additionally, to increase useracceptability, drug delivered to the vagina should be retained in thevaginal tract at effective concentrations over extended periods of time.Achieving sustained local drug concentrations is challenging because thevaginal epithelium is highly permeable to small molecules and alsobecause soluble drug dosage forms (gels, creams) can be expelled byintra-abdominal pressure and ambulation. Lastly, drug delivery methodsmust be safe and non-toxic to the vaginal epithelium. Improvements inthe distribution, retention, and safety profile of vaginal dosage formsmay lead to a substantial increase in efficacy and decrease in the sideeffects caused by largely ineffective systemic treatments forcervicovaginal infections and diseases.

Nanoparticles have received considerable attention owing to theirability to provide sustained local drug delivery to the vagina. However,the mucus layer coating the vaginal epithelium presents a barrier toachieving uniform distribution and prolonged retention in the vaginaltract. Mucus efficiently traps most particulates, including conventionalpolymeric nanoparticles (CPs), through both adhesive and stericinteractions. The efficiency with which mucus traps foreign pathogensand particulates implies that CPs would become trapped immediately uponcontact with the lumenal mucus layer, preventing penetration into and,thus protection of, the rugae. Particles and pathogens trapped in thesuperficial lumenal mucus layer would be expected to be rapidly clearedfrom the tissue, limiting the retention time of mucoadhesive materials,such as CP.

By mimicking viruses that have evolved to penetrate the mucus barrier toestablish infection, mucus-penetrating particles (MPPs) were recentlyengineered for mucosal drug delivery by coating CPs with anexceptionally high density of low molecular weight poly(ethylene glycol)(PEG). MPPs diffuse through human cervicovaginal mucus (CVM) at speedscomparable to their theoretical diffusion through water. Here, it wassought to test the hypothesis that MPPs would provide enhanceddistribution and increased retention in vivo in the vagina bypenetrating into the deepest mucus layers, including the more slowlycleared mucus in the rugae, thereby releasing drug in the optimallocation for efficient tissue uptake (FIG. 14E). In addition to thecommon progestin-induced diestrus phase (DP) mouse model, the use of anestradiol-induced estrus phase (IE) mouse model is introduced in whichthe mouse CVM (mCVM) more closely mimics human CVM (hCVM) and,therefore, provides a more human-like model for developing andtranslating MPPs for human use.

Carboxylic acid-coated, fluorescent polystyrene nanoparticles (PS-COOH)were made into MPPs by covalently attaching a dense coating of lowmolecular weight PEG, as previously reported (Wang Y Y et al. Angew ChemInt Ed Engl 47:9726-9729, 2008; Lai S K et al. Proc Natl Acad Sci USA104:1482-1487, 2007). Additionally, biodegradable MPPs (BD-MPP) wereformulated with a poly(lactic-co-glycolic acid) (PLGA) core and aphysically adsorbed PEG coating, as previously reported (Yang M et al.Angew Chem Int Ed Engl 50:2597-2600, 2011), because biodegradableparticles can be loaded with drugs and are suitable for dosing tohumans. PS-COOH and PLGA nanoparticles have a highly negative surfacecharge, which is nearly neutralized when densely coated with PEG.Nanoparticles were determined to be well-coated by measuring zetapotential (Table 12) as described previously (Lai S K et al. 2007). Azeta potential more neutral than −10 mV was previously found to benecessary for mucus-penetrating properties in hCVM (Wang Y Y et al,2008). To ensure that MPPs were mucus-penetrating in native estrus phasemCVM, the particles were administered intravaginally to mice in theestrus phase. The entire vagina was then excised and opened to visualizethe motions of hundreds of individual particles with a multiple particletracking (MPT) method (Suh J et al. Adv Drug Deliv Rev 57:63-78, 2005).Particle trajectories for MPPs were indicative of rapid diffusionthrough watery pores in the mCVM, whereas motions of uncoated PS-COOHnanoparticles (CPs) were smaller than the particle diameter (˜100 nm)(FIG. 14A). The ensemble averaged mean squared displacement (<MSD>) ofMPPs in mCVM was found to be comparable to that reported for MPPs inhCVM (Lai S K et al. Proc Natl Acad Sci USA 107:598-603, 2010) (FIG.14B), corresponding to ensemble averaged effective diffusivity (<Deff>)only ˜8-fold slower than the theoretical diffusion of 110 nm particlesin water (˜4 μm²/s).

TABLE 12 Particle characterization. Size, zeta potential, andpolydispersity index (PDI) for all particle formulations. Particle typeSize (nm) ζ potential (mV) PDI PSPEG (MPP) 112 ± 3 −4.2 ± 0.6 0.03 PS(CP)  87 ± 4 −39.0 ± 2.4  0.10 PLGA/F127 (BD-MPP) 152 ± 6 −4.2 ± 0.30.06 PLGA/PVA (BD-CP)  161 ± 15 −6.7 ± 0.1 0.06 ACVp-MPP  65.3 ± 10 −6.3± 1.0 0.37

Based on the measured D_(eff) for individual particles, it was estimatedwith Fick's Second Law of Diffusion that about half of the MPP woulddiffuse through a 100 μm-thick layer of mCVM in about 4 h (FIG. 14D),whereas even after 24 h there would be no appreciable penetration byCPs. D_(eff) values for CPs at a time scale of 1 s corresponded to MSDvalues less than the particle diameter (dotted line, FIG. 14C), likelyrevealing thermal fluctuation of particles stuck to mucin fibers and notparticle diffusion. Overall, the transport behavior of both MPPs and CPsin estrus phase mCVM was very similar to their transport behavior inhCVM.

Synchronizing a large number of mice in the estrus phase for retentionstudies required hormonal treatment. Particle transport behavior wastested in IE mice to confirm that estradiol treatment, which has beenused routinely for inducing estrus-like behavior in many animal models(Ring J R, Endocrinology 34:269-275, 1944; Rubio C A, Anat Rec185:359-372, 1976), did not alter MPP and CP transport behavior prior todistribution and retention studies (FIG. 15A). Additionally, BD-MPPtransport behavior was indistinguishable from MPP in IE mucus (FIG.15B).

It was next investigated in the estrus phase mouse and IE mouse whetherthe ability to rapidly penetrate mucus would lead to more rapid anduniform vaginal distribution of MPPs compared to CPs. MPPs and CPs wereapplied in hypotonic media to mimic the way osmotically driven waterflux (advective transport) rapidly transports nutrients from theintestinal lumen to the brush border epithelial surface. Ten minutesafter particle administration, the entire vagina was dissected out andstained for cell nuclei. CPs aggregated in the lumenal mucus and did notpenetrate into the vaginal rugae (FIG. 16). In contrast, MPPs formed acontinuous particle layer that coated the entire vaginal epithelium,including all the surfaces of the rugae. MPPs penetrated more than ˜100μm of mucus via advection within 10 min compared to the ˜4 hours itwould take them to diffuse that distance through mucus (FIG. 14D). Thisbehavior was also consistent for BD-CPs and BD-MPPs, and CPs and MPPsadministered to IE mice (FIG. 16). Videos illustrating the movement ofMPPs through hCVM past muco-adhesive CPs can be found in Video 1 (noflow, diffusion) and Video 2 (with flow, advection).

To quantify the difference in distribution of MPPs and CPs, fluorescentimages were obtained of freshly excised, opened, and flattened mousevaginal tissue. As can be seen in FIG. 17, the adhesion of CPs tolumenal vaginal mucus layers created “stripes” of mucus with particlesalternating with dark “stripes” of mucus without particles, the lattercorresponding to the rugae that were opened when the vaginal tissue wasflattened. In contrast, transport of MPPs toward the epithelium and intothe rugae created a continuous particle coating on the flattened vaginalsurface (FIG. 17). Quantification of the fluorescence on the vaginal andcervical tissue indicated that 88% of the flattened vaginal surface and87% of the cervical surface were densely coated with MPPs, whereas only30% of the vaginal surface and 36% of the cervical surface were coatedwith CPs. Upon further inspection at higher magnification of darkerareas of the vaginal and cervical surfaces, a continuousless-concentrated coating of MPPs was seen (FIGS. 17-18, insets),implying that there was nearly complete coverage of the vaginal andcervical epithelium. For CPs, a less-concentrated coating was not foundat higher magnification (FIGS. 17-18, insets). Similar trends were foundwith BD-MPPs, with 85% vaginal coverage and 86% cervical coverage, aswell as BD-CPs with 31% vaginal coverage and 27% cervical coverage(FIGS. 17-18).

It was then sought to determine whether the improved distribution ofBD-MPP could improve the delivery of small molecules as compared to agel dosage form. Lipophilic molecules are likely to enter the firstepithelial surface they contact, failing to contact cells in the rugae.Conversely, hydrophilic molecules can diffuse rapidly through thevaginal epithelium and be carried away by blood and lymph circulationleading to brief periods of coverage. BD-MPP was loaded with afluorescent, water-soluble small molecule, fluorescein isothiocyanate(FITC), as a model drug (FITC/MPP). To mimic conventional vaginaldelivery, soluble FITC (FITC/gel) was administered in the universalvaginal placebo gel hydroxyethylcellulose (HEC). Twenty-four hours afteradministration to estrus phase mice, the vaginal tissues were excisedand flattened to expose the vaginal folds. Patches of FITC coated 42% ofthe vaginal surface when administered as FITC/gel, whereas FITC/MPPprovided a well-retained FITC coating of 87% of the vaginal surface(FIG. 22), even 24 h after particle administration.

To further characterize the effects of the mucus barrier, we found thatremoving vaginal mucus by lavage (Cu Y et al., J Cont Rel 156:258-64,2011; Woodrow K A et al. Nat Mater 8:526-33, 2009) prior to particleadministration markedly improved CP distribution, indicating that theirmucoadhesive character prevents uniform distribution in the vagina (FIG.19).

It was next sought to determine vaginal retention of MPPs compared tomucoadhesive CPs using our IE model. Fluorescent MPPs and CPs wereadministered intravaginally to IE mice. At specified time points, theentire reproductive tract (vagina and uterine horns) was excised andanalyzed quantitatively with fluorescence imaging (FIG. 21A). After aninitial decrease in particle fluorescence that was similar for MPP andCP (likely owing to initial “squeeze out” preceding mucus penetration),the remaining amount of MPPs stayed constant at roughly 60% (FIG. 21B).In contrast, the amount of CPs steadily decreased with time to 10% (6h). Importantly, although CPs were distributed along the length of thevagina, this longitudinal coverage does not indicate that the CPpenetrated mucus to reach the epithelium, nor surfaces inside thevaginal folds, as shown in FIG. 16.

The immune system is highly active at mucosal surfaces (Mestecky O P etal. Mucosal Immunology. Elselvier Academic Press, Burlington, ed. Third,2005), especially those with surfaces covered with living cells, such ascolumnar epithelia in the endocervix in humans. Inflammatory effects ofnanoparticles were investigated using mice pre-treated withDepo-Provera, a long-acting progestin treatment that synchronizes micein the diestrus phase, during which the vaginal epithelium thins andbecomes covered with living cells. In contrast, in estrus, the mousevagina thickens from 4 to 7 cell layers to about 12 cell layers and theepithelial surface is protected with many layers of dead and dying cells(Biology of the laboratory mouse. Ed. G D Snell. Dover Publications,Inc., New York, 1956; J Am Pharml Assoc 45:819-819, 1956). Additionally,the progestin-induced diestrus phase (DP) mouse vaginal epithelium hasan increased immune cell population, leading to enhanced acuteinflammatory responses; whereas the estrus phase is characterized by anabsence of immune cells (Hubscher C H et al. Biotech Histochem 80:79-87,2005). Depo-Provera effectively synchronizes mice to a diestrus-likestate for days to weeks, which is important for experiments lasting 24 hor longer.

Standard hematoxylin and eosin (H&E) staining was used to investigatepotential toxic effects of intravaginally administered nanoparticles.Nonoxynol-9 (N9), a nonionic detergent known to cause vaginal toxicity(Ramjee G et al. AIDS 24 Suppl 4, S40-49. 2010), was used as a positivecontrol, and PBS (saline) was used as a negative control. The same (BD-)MPPs and (BD-) CPs that were used for distribution and retention studieswere tested for toxicity. As expected, N9 caused acute inflammation at24 h that was not seen following PBS treatment (FIG. 23). CP, like N9,caused pronounced neutrophil infiltration into the lumen, but MPPs didnot cause this inflammatory effect (FIG. 23, arrowheads).

Recent studies indicate that in response to certain vaginal products,the vaginal epithelium can secrete immune mediators that may enhancesusceptibility to sexually transmitted infections (Cummins J E et al.Sex Transm Dis 36:S84-91, 2009; Wilson S S et al. Antivir Ther14:1113-1124, 2009). Thus, it is important that a vaginal product notinduce such an immune response, particularly after repeated dosing.Because our ultimate goal was to test MPPs for protection against HSV-2,we compared an MPP formulation containing acyclovir monophosphate (ACVp)to N9, HEC placebo gel, PBS, and a gel vehicle (TFV vehicle) used inrecent tenofovir clinical trials. Nanoparticle and control formulationswere administered vaginally to Depo Provera-treated mice daily for sevendays. Vaginal lavages were collected on day 8 from each mouse andassessed for cytokines that have been found to be elevated in responseto epithelial irritation: interleukin 1β (IL-1β), interleukin 1α(IL-1α), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6). Itwas found that both IL-1α and IL-1β levels were elevated in response toboth the TFV vehicle and N9 solution (FIG. 24). This was not surprisingin the case of N9 treatment, considering IL-1α and IL-1β are secreted bythe vaginal epithelium in response in injury (Cummins J E et al., 2009).In contrast, the cytokine levels associated with ACVp-MPP wereequivalent to the levels associated with HEC placebo gel (FIG. 24),which has been used in clinical trials without any associated increasein susceptibility to infection (Karim Q A et al. Science 329:1168-1174,2010; Tien D et al. AIDS Res Hum Retroviruses 21:845-853, 2005). Therewas no detectable elevation of either IL-6 or TNF-α associated with anyvaginal treatment as compared to untreated controls.

It was finally investigated whether the improved distribution,retention, and toxicity profile of MPPs would lead to improvedprotection against vaginal HSV-2 challenge in mice. Depo Proveratreatment markedly increases the vaginal susceptibility of mice toinfections, and candidate microbicides have provided only partialprotection in the mouse model used here, even when administeredimmediately before the infectious inoculum (Achilles S L et al. SexTransm Dis 29:655-664; 2002; Zeitlin L et al. Contraception 56:329-335,1997). Moreover, several vaginal product excipients actually increasesusceptibility to infection in this model (Cone R A et al., 2006; MoenchT R et al. BMC Infect Dis 10:331, 2010). It was chosen to test ACVp forblocking vaginal transmission of HSV-2 infections, because acyclovirprovides viral suppression in animals with repeated dosing multipletimes per day (Kern E R, Am J Med 73, 100-108 (1982)). However, a singlevaginal pretreatment with 50 mg/mL (5%) ACVp in guinea pigs resulted in70% of animals infected compared to controls (Kern et al. NucleosNucleot Nucl 19:501-513, 2000). Therefore, ACVp provided a test case todetermine whether MPP could significantly improve protection by awater-soluble and quickly metabolized drug by prolonging therapeuticallyrelevant drug concentrations after a single application. Additionally,the mechanism of action of nucleotide analogs, such as ACVp, isprevention of intracellular viral replication, such that successfulprotection implies efficient uptake and retention in susceptible targetcell populations in the vaginal and cervical mucosa.

ACVp nanoparticles were formulated with the same muco-inert coating usedfor all other studies. The size and ξ-potential of ACVp-MPP were similarto polystyrene (PS)-based MPP (Table 12). Mice were administered solubleACVp or ACVp-MPP intravaginally 30 min prior to HSV-2 challenge. Solubledrug administered at the same concentration as the ACVp-MPP (1 mg/mL)was ineffective at protecting mice from viral infection (84.0% infectedcompared to 88.0% of controls), whereas only 46.7% of mice in theACVp-MPP group were infected (Table 11). Groups of mice given solubledrug at 10-times the concentration in ACVp-MPPs were still infected at arate of 62.0% (drug in PBS) or 69.3% (drug in water). Comparing solubledrug to ACVp-MPP in the same vehicle (pure water), soluble drug wassignificantly less protective, even at 10-fold higher concentration thanACVp-MPP (Table 11). To study the distribution and retention ofnanoparticles at the vaginal mucosal surface and the effects of repeateddosing, 6-8 week old CF-1 mice (Harlan) were used. Mice were housed in areversed light cycle facility (12 h light/12 h dark). For naturallycycling estrus, mice were selected for external estrus appearance andconfirmed upon dissection (Champlin A K et al. Biol Reprod 8: 491-494,1973; Allen E, Am J Anatomy 30:297-371, 1922). For hormonally inducedestrus (IE), mice were acclimated for 3 weeks and injectedsubcutaneously with 100 μg of 17-13 estradiol benzoate (Sigma) two daysprior to the experiments. It has been demonstrated in numerous studiesthat treatment with estradiol induces an “estrus-like” state withanalogous epithelial characteristics and vaginal cell populations (RosaC G et al. Ann N Y Acad Sci 83:122-144, 1959; Rubio C A, Anat Rec185:359-372, 1976; Gillgrass et al. J Virol 79:3107-3116, 2005). Forvaginal toxicity and cytokine release, mice were injected subcutaneouslywith 2.5 mg of Depo-Provera (medroxyprogesterone acetate, 150 mg/mL)(Pharmacia & Upjohn Company) 7 days prior to the experiments.

Water was used as the hypotonic medium for all particle solutions. Forex vivo tracking, L of particles were administered intravaginally. Afterapproximately 10 min, the vagina was removed and carefully sliced opento lay flat. The whole tissue was placed in a custom-madewell-constructed such that a cover slip could be placed on top tocontact the mucus without deforming the tissue. The well was a rectangleapproximately 1 mm×0.5 mm cut out of three layers of electrical tapeadhered to a standard glass slide. Cover slips were sealed around theedges with superglue and imaged immediately to prevent drying.

Mice were anesthetized prior to experimental procedures, includingsacrifice by cervical dislocation. For all studies, mice were preventedfrom self-grooming by a collar of mildly adhesive tape around theabdomen, and from inter-grooming by housing in individual cages.

For conventional mucoadhesive particles (CPs), fluorescent, carboxyl(COOH)-modified polystyrene (PS) nanoparticles sized 100 nm (MolecularProbes) were used. These particles feature a negatively charged surfaceat neutral pH (Table 12). To produce mucus-penetrating particles (MPPs),CPs were covalently modified with 5-kDa amine-modified PEG (CreativePEGworks) via standard 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidecoupling reaction. Particle size and ξ-potential were determined bydynamic light scattering and laser Doppler anemometry, respectively,using a Zetasizer Nano ZS90 (Malvern Instruments). Size measurementswere performed at 25° C. at a scattering angle of 90°. Samples werediluted in 10 mM NaCl solution (pH 7) and measurements performedaccording to instrument instructions. A near-neutral ξ-potential,measured by laser Doppler anemometry, was used to confirm PEGconjugation.

For biodegradable particles, PLGA acid 2A (50:50 LakeshoreBiomaterials), Lutrol F127 (BASF), and poly(vinyl alcohol) (PVA 25 kDa,Polysciences) were used. Alexa Fluor 555 was chemically conjugated toPLGA, which was used to produce nanoparticles by nanoprecipitation asdescribed previously (Yang M et al. Angew Chem Int Ed Engl 50:2597-2600,2011). Briefly, 10 mg/mL of labeled PLGA was dissolved in acetone or THF(with or without 2 mg FITC), and added dropwise to 40 mL of aqueoussurfactant solution. After stirring for 2 h, particles were filteredthrough a 5-μm syringe filter and collected by centrifugation (SorvallRC-6+, ThermoScientific) and washed. Particle size and ξ-potential weredetermined as described.

ACVp-MPPs were prepared by dissolving ACVp in ultrapure water containingLutrol F127. Zinc acetate was added at a molar ratio of 5:1 ACVp:Zn, tochelate the ACVp and render it water insoluble, and then immediatelyflash frozen and lyophilized. Particle characterization was conductedafter reconstitution. The powder was reconstituted with ultrapure waterprior to administration, with a final concentration of 1 mg/mL ACVp and0.8 mg/mL Lutrol. Soluble ACVp was titrated with NaOH as needed to reachpH to 6-7. Particle size and ξ-potential were determined as described.

The trajectories of the fluorescent particles in ex vivo vaginal tissuesamples were recorded using a silicon-intensified target camera(VE-1000, Dage-MTI) mounted on an inverted epifluorescence microscopeequipped with 100× oil-immersion objective (numerical aperture 1.3).Movies were captured with Metamorph software (Universal Imaging Corp.)at a temporal resolution of 66.7 ms for 20 s. Trajectories of n≥130particles were analyzed for each experiment, and three independentexperiments were performed using tissue from different mice. Thecoordinates of particle centroids were transformed into time-averagedmean squared displacements (<MSD>), calculated as:<Δr ²(τ)>=[x(t+τ)−x(t)]²+[y(t+τ)−y(t)]²where τ is time scale (or time lag), x and y are the correspondingparticle coordinates at time t, and Δr² is the MSD. This equation wasused to calculate particle MSDs and effective diffusivities (D_(eff)),as previously demonstrated (Lai S K et al. Proc Natl Acad Sci USA104:1482-1487, 2007; Tang B C et al. Proc Natl Acad Sci USA106:19268-19273, 2009). The calculated D_(eff) values were used formodeling of particle penetration through a mucus slab, as describedpreviously (Tang B C et al, 2009).

For distribution with mucus removal, prior to particle administration,mice were given 2× vaginal lavage with 50 μL of PBS followed by a singleswab with a cotton-tipped applicator. Subsequently, 5 μL of either CPsor MPPs were administered intravaginally. The entire vagina was thenremoved and frozen in Tissue-Tek O.C.T. Compound (Sakura Finetek U.S.A.,Inc.). Transverse sections were obtained at various points along thelength of the tissue (between the introitus and the cervix) using aMicrom HM 500 M Cryostat (Microm International). The thickness of thesections was set to 6 μm to achieve single cell layer thickness. Thesections were then stained with ProLong Gold (Invitrogen) antifadereagent with DAPI to visualize cell nuclei and retain particlefluorescence. Fluorescent images of the sections were obtained with aninverted fluorescent microscope. To quantify nanoparticle distribution,5 μL of either CPs or MPPs were administered intravaginally. Within 10minutes, vaginal tissues, including a “blank” tissue with no particlesadministered, were sliced open longitudinally and clamped between twoglass slides sealed shut with super glue. This procedure completelyflattens the tissue, exposing the folds. The “blank” tissue was used toassess background tissue fluorescence levels to ensure that all imagestaken were well above background levels. Six fluorescent images at lowmagnification and at least one image at high magnification were takenfor each tissue. The images were thresholded to draw boundaries aroundthe fluorescent signal, and then the area covered quantified usingImageJ software. An average coverage was determined for each mouse, andthen these values were averaged over a group of n≥3 mice. The cervixfrom each mouse was cut from the uterine horns and mounted using thesame custom-made wells used for ex vivo particle tracking. The wellswere sealed with a cover slip, and the background fluorescence levelsdetermined using the blank tissue. One fluorescent image, constitutingnearly the entire cervical surface, was taken at low magnification abovetissue background levels. These images were thresholded in the samemanner to determine the area covered with particles. At least one highermagnification image was taken for each tissue to show individualparticles.

Advection of MPPs and CPs were visualized using a custom capillary tubesetup. A flat capillary tube (0.4 mm×4 mm×50 mm; VitroCom) was attachedto a 1 mL tuberculin syringe (Becton Dickinson) via a piece of flexibleplastic tubing. The tubing was clamped to one end of the capillary tube,and sealed using silicone grease. The syringe and tubing were loadedwith saline, followed by fresh, undiluted human CVM mixed with 3% (v/v)of ˜500 nm uncoated (red fluorescent) and PEG-coated (green fluorescent)polystyrene beads (Invitrogen). PEG-coated beads were prepared asdescribed above for the 100 nm MPP used in mouse studies. Approximately80 μL of mucus was needed to fill the capillary tube, and care was takento avoid introducing air bubbles. Time lapse videos showing the motionsof MPP and CP within the capillary tube, with and without appliedpressure, were recorded using a 40× objective on a Zeiss LSM 510confocal microscope (Carl Zeiss MicroImaging, LLC).

FITC dye (Sigma-Aldrich) was mixed at 1 mg/mL in HEC gel kindly providedby T. Moench (Reprotect). Biodegradable MPP were prepared as described,loaded with FITC dye and suspended in 1% Lutrol F127. To evaluatedistribution, 10 μL of either gel or particle solution was administeredintravaginally. After 24 h, the vaginal tissue was removed and cut opento lie flat. The tissue was then mounted between two microscope slidesand squeezed to flatten the rugae. A “blank” tissue was included todetermine background autofluorescence from the vaginal tissue, to ensurethat the exposure setting used was indicative of FITC presence.Fluorescent images of the dye distribution on the flattened tissuesurface were obtained using a Nikon E600 inverted microscope equippedwith a 2× objective. These images were thresholded in the same mannerusing ImageJ to determine the coverage area.

To evaluate nanoparticle retention, 5 μL of red fluorescent CPs or MPPswere administered intravaginally. Whole cervicovaginal tracts wereobtained at 0, 2, 4, and 6 h and placed in a standard tissue culturedish. For each condition and time point, at n>7 mice were used.Fluorescence images of the tissues were obtained using the Xenogen IVISSpectrum imaging device (Caliper Life Sciences). Quantification offluorescent counts per unit area was calculated using the Xenogen LivingImage 2.5 software.

Five μL of particles or control solutions were administeredintravaginally to the DP mouse model. After 24 h, whole cervicovaginaltracts were obtained and fixed in 4% paraformaldehyde solution for 24 h.Tissues were placed in 70% ethanol and taken to the Johns HopkinsReference Histology Laboratory for paraffin embedding and standard H&Estaining.

Twenty μL of each test agent was administered intravaginally to the DPmouse model once-a-day for seven (7) days. HEC gel and N9 were providedby T. Moench (Reprotect), and TFV vehicle gel was kindly provided by C.Dezzutti (University of Pittsburgh). On the eighth day, each mouse waslavaged twice with 50 μL of PBS. Each lavage sample was diluted with anadditional 200 μL of PBS and centrifuged to remove the mucus plug.Supernatant (200 μL) was removed and split into 50 μL for each of thefour (IL-1β, IL-1α, TNF-α, and IL-6) Quantikine ELISA kits (R&D Systems,Inc.). ELISAs were conducted per the manufacturer's instructions.

All data are presented as a mean with standard error of the mean (SEM)indicated. Statistical significance was determined by a two-tailed,Student's t-test (α=0.05) assuming unequal variance. In the case ofHSV-2 challenge, statistical significance was determined using Fisher'sexact test, two-tailed distribution.

The female reproductive tract is susceptible to a wide range of sexuallytransmitted infections (R. Mallipeddi, L. C. Rohan, Expert Opin DrugDeliv 7:37-48, 2010). Biological vulnerability, a lack offemale-controlled prevention methods, and inability to negotiate condomuse all contribute to male-to-female transmission worldwide (Mallipeddi,2010; Ndesendo, V M et al. AAPS PharmSciTech 9:505-520, 2008). An easilyadministered, discreet, and effective method for protecting womenagainst vaginal HIV, HSV-2, and other virus transmission could preventmillions of infections worldwide. After 11 unsuccessful microbicidetrials, CAPRISA 004 was the first to demonstrate partial protectionagainst HIV with a vaginally administered microbicide (tenofovir) in agel formulation (Karim Q A et al. Science 329:1168-1174, 2010). Animportant difference between previous generation microbicides such as N9and the current generation of microbicides is the site of action. Manycurrent generation microbicides, such as nucleotide analogs tenofovirand acyclovir monophosphate, work intracellularly to inhibit viralreplication, whereas previous generations directly inactivated pathogensin the vaginal lumen. However, some previous generation microbicidescaused toxicity to the vaginal epithelium that increased susceptibilityto infection (D'Cruz O J et al. J Antimicrob Chemo 57:411-423, 2006).

For vaginal drug delivery to be maximally effective, topically delivereddrugs must be distributed uniformly, maintained at sufficiently highconcentration, and remain in close proximity to the folded vaginalepithelium (rugae) and cervical mucosa. Several techniques have beenused to observe distribution of gels and drugs following vaginaladministration, such as MRI (Mauck C K et al. Contraception 77:195-204,2008), gamma-scintigraphy (Mauck C K et al., 2008; Chatterton B E et al.Int J Pharm 271:137-143, 2004), colposcopy (Poelvoorde N et al. Eur JPharm Biopharm 73:280-284, 2009), and fiber optics (Mauck C K et al,2008). These techniques are adequate to observe gross distribution alongthe vaginal tract, but do not reveal entry into vaginal folds. Our workdemonstrates that, although a topical treatment may be well-distributedlongitudinally along the vaginal tract, much of the folded epitheliumcan be left untreated and unprotected. Such untreated surfaces couldhave contributed to recent failures of several candidate microbicidesagainst HIV in clinical trials (Hendrix C W et al. Annu Rev PharmacolToxicol 49:349-375, 2009). Additionally, when a fluid or gel isadministered to the vagina, it directly contacts the rapidly shed outerlumenal mucus layer. Mucoadhesive particles, such as CP, are trapped inthis superficial mucus layer and thereby excluded from the rugae. Incontrast, we demonstrated that MPPs are capable of penetrating deep intothe mouse rugae and, when delivered hypotonically, provided completecoverage of the epithelium within only 10 minutes.

Diffusion of particles is not rapid enough to result in such a uniformepithelial coating within minutes. Diffusion over ˜100 μm would take onthe order of hours. However, the vaginal epithelium has a great capacityfor fluid absorption induced by osmotic gradients. Absorption of waterthrough the mucus barrier assists MPPs in rapidly reaching the entireepithelial surface by advection, where the drug payload can then bereleased for optimal tissue uptake. In contrast, water absorption wasnot beneficial for CP, because they became adhesively trapped andimmobilized in the lumenal mucus (Video 2).

Inadequate retention of therapeutically active compounds in the vaginaltract is another limiting factor for vaginal protection. For example,many vaginal spermicides provide protection for no more than 1 h(Zaneveld L J D et al. J Androl 22:481-490, 2001). Other vaginalproducts are not well-retained even after 6 h (Omar R F et al.Contraception 77:447-455, 2008; N. Poelvoorde, 2009; Chatterton B E etal. Int J Pharm 271:137-143, 2004), necessitating repeatedadministration for adequate protection. Similarly, over 90% of CPs wereshed from the vagina within 6 h because they did not penetrate deep intothe mucus layers. In contrast, MPPs provided enhanced delivery of anencapsulated model drug (FITC) for at least 24 h, as compared to solubledrug in a gel formulation. Thus, MPPs may provide a means for achievingpotent once-daily topical vaginal administration for treatments such asmicrobicides against sexually transmitted diseases.

In prior attempts to develop mucosal drug delivery systems for thevaginal tract, a variety of “pretreatments” have been used that diminishthe mucus barrier. Administering fluids (Cu Y et al. J Control Release,(10.1016/j.jconrel.2011.06.036); Woodrow, K A et al. Nat Mater8:526-533, 2009; Kanazawa T et al. Int J Pharm 360:164-170, 2008;Kanazawa T et al. J Pharm Pharmacol 61:1457-1463, 2009), swabs (WoodrowK A et al, 2008; Kask As et al. Vaccine 28:7483-7491, 2010), ordegradative enzymes (Seavey M M et al. Vaccine 27:2342-2349, 2009) priorto administration of mucoadhesive delivery vehicles was likely essentialto the drug or gene delivery achieved in these studies. Here, it wasfound that a lavage plus swab pretreatment markedly improveddistribution of CPs in the vagina, allowing the particles to coat theepithelium similarly to MPP (FIG. 19). Barrier-removing pretreatmentsmay be impractical for human use, and especially inappropriate formicrobicides intended to prevent sexually transmitted diseases. HealthyCVM itself is a somewhat effective barrier to viral infections (Lai S Ket al. J Virol 83:11196-11200, 2009). It was shown that effectiveepithelial coverage can be achieved by using MPPs without the need todegrade or remove the mucus barrier.

PEG coatings have been widely used in developing polymeric drug carriersthat are not easily recognized by the immune system (Tang B C et al.Proc Natl Acad Sci USA 106: 19268-73, 2009). It was demonstrated thatdense PEG coatings produce MPPs that rapidly penetrate mucus withoutcausing inflammation in the mouse vaginal tract. In contrast,administration of uncoated CPs resulted in an acute inflammatoryresponse similar to administration of N9. Additionally, cytokine levelsassociated with daily administration of MPPs were indistinguishable fromHEC placebo gel. Elevated levels of IL-1α and IL-1β, which areassociated with epithelial injury, occurred after daily dosing with bothN9 and TFV vehicle gel. The tenofovir-containing version of this gel wasshown to have complete protection against HIV in a tissue explant model,and complete protection occurred in spite of visible epithelial shedding(Rohan L C et al. PLoS One 5:e9310, 2010). Previous work suggests thatglycerol in the TFV gel may be responsible for the observed toxicity inmice (Moench T R et al. BMC Infect Dis 10:331, 2010).

Mice are useful animal models for developing vaginal products, but thereare key differences in vaginal physiology between mice and humans.First, the estrous cycle occurs over a 4 to 5 day period in contrast tothe 28-day human menstrual cycle. Throughout the four stages of themouse estrous cycle, substantial growth is followed by sloughing of theepithelium, whereas there is relatively little change in the humanvaginal epithelium throughout the menstrual cycle (Smith B G et al. Am JAnat 54:27-85, 1934; Ildgruben A K et al. Obstet Gynecol 102:571-582,2003). The late proestrus and early estrus phases of the mouse estrouscycle are the most similar to that of the human vaginal epithelium(Smith B G et al., 1934; Asscher A W et al. J Anat 90:547-552, 1956). Inthese stages, there is significant bacterial colonization, including apeak in the presence of lactobacilli (Cowley H M et al. Microb EcolHealth Dis 4:229-235 1991). Additionally, the estradiol influence causesactive secretion of mucus (Cowley H M et al., 1991; Corbeil L B et al.Tissue Cell 17:53-68, 1985; Rosa C G et al. Ann N Y Acad Sci 83:122-144,1959), which was found in mice is both penetrable by MPPs and cleared ina matter of hours, similar to humans. Thus, it is believed that the IEmouse model is a valuable model in addition to the commonly used DPmodel for investigating vaginal delivery methods. Estradiol can be usedto synchronize mice in the estrus phase, but does not “arrest” them inestrus. They continue to cycle, whereas DP treatment can arrest mice ina diestrus-like phase for days to weeks (Kaushic C et al. J Virol77:4558-4565, 2003).

Is has been shown that MPPs are capable of rapidly penetrating humancervicovaginal and mouse vaginal mucus and that MPPs significantlyimprove speed and uniformity of coverage and retention time compared toconventional mucoadhesive nanoparticles. CPs elicited acute inflammatoryresponses similar to a known irritant N9, but, similar to the placebogel, MPP caused no detected inflammatory responses. Vaginallyadministered MPPs loaded with acyclovir monophosphate were moreeffective at protecting mice against vaginal HSV-2 infection thansoluble drug, even at 10-fold higher soluble drug concentration. Theseresults motivate further development of MPPs for safe and effectivevaginal drug delivery, for prevention and treatment of sexuallytransmitted infections, contraception, and treatment of othercervicovaginal disorders.

Other Embodiments

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

The invention claimed is:
 1. A mucus-penetrating anti-microbialcomposition for respiratory delivery, the composition comprising aplurality of coated particles, wherein each of the coated particlescomprise: a core particle comprising an anti-microbial agent, whereinthe anti-microbial agent constitutes at least 80 wt % of the coreparticle; and a coating comprising a surface-altering agent surroundingthe core particle, wherein the surface-altering agent comprises atriblock copolymer comprising a hydrophilic block—hydrophobicblock—hydrophilic block configuration, wherein the hydrophobic block hasa molecular weight of at least 2 kDa, and the hydrophilic blocksconstitute at least 15 wt % of the triblock copolymer, and wherein thehydrophobic block associates with the surface of the core particle andrenders the coated particle hydrophilic; and wherein the coatedparticles have an average size of at least 5 nm and less than or equalto 1000 nm.
 2. The composition of claim 1, wherein the triblockcopolymer is poly(ethylene oxide)-poly(propylene oxide)-poly(ethyleneoxide) or poly(ethylene glycol)-poly(propylene oxide)-poly(ethyleneglycol).
 3. The composition of claim 2, wherein the poly(ethylene oxide)or poly(ethylene glycol) block has a molecular weight of at least 2 kDa.4. The composition of claim 1, wherein the coated particles have anaverage size of at least 50 nm and less than or equal to 500 nm.
 5. Thecomposition of claim 1, wherein the coated particles have an averagesize of less than or equal to 400 nm.
 6. The composition of claim 1,wherein the coated particles diffuse through human cervicovaginal mucusat a diffusivity that is greater than 1/500 the diffusivity that theparticles diffuse through water on a time scale of 1 second.
 7. Thecomposition of claim 1, wherein the coated particles have a relativevelocity of greater than 0.5 in mucus.
 8. The composition of claim 1,wherein the anti-microbial agent is an anti-bacterial agent.
 9. Thecomposition of claim 8, wherein the anti-bacterial agent is penicillin,benethamine penicillin, cinoxacin, ciprofloxacin, clarithromycin,clofazimine, cloxacillin, demeclocycline, doxycycline, erythromycin,ethionamide, imipenem, nalidixic acid, nitrofurantoin, rifampicin,spiramycin, sulphabenzamide, sulphadoxine, sulphamerazine,sulphacetamide, sulphadiazine, sulphafurazole, sulphamethoxazole,sulphapyridine, tetracycline, or trimethoprim.
 10. The composition ofclaim 1, wherein the anti-microbial agent is an anti-viral agent. 11.The composition of claim 10, wherein the antiviral agent is acyclovir ortenofovir.
 12. The composition of claim 1, wherein the anti-microbialagent is an anti-fungal agent.
 13. The composition of claim 12, whereinthe anti-fungal agent is amphotericin, butoconazole nitrate,clotrimazole, econazole nitrate, fluconazole, flucytosine, griseofulvin,itraconazole, ketoconazole, miconazole, natamycin, nystatin, sulconazolenitrate, terbinafine HCl, terconazole, or tioconazole.
 14. A method ofdelivering an anti-microbial agent across a mucosal barrier of a tissuein the respiratory system, the method comprising delivering to themucosal barrier the anti-microbial composition of claim
 1. 15. Themethod of claim 14, wherein the mucosal barrier is mucus or a mucosalmembrane.
 16. The method of claim 14, wherein the mucosal barrier ispresent in a mucosal tissue.
 17. The method of claim 14, wherein thetissue is lung, nasal, pharyngeal, tracheal, or bronchial tissue. 18.The method of claim 14, wherein the anti-microbial composition isadministered by inhalation, nasal spray, or any topical administration.19. A mucus-penetrating anti-microbial composition for gastrointestinaldelivery, the composition comprising a plurality of coated particles,wherein each of the coated particles comprise: a core particlecomprising an anti-microbial agent, wherein the anti-microbial agentconstitutes at least 80 wt % of the core particle; and a coatingcomprising a surface-altering agent surrounding the core particle,wherein the surface-altering agent comprises a triblock copolymercomprising a hydrophilic block—hydrophobic block—hydrophilic blockconfiguration, wherein the hydrophobic block has a molecular weight ofat least 2 kDa, and the hydrophilic blocks constitute at least 15 wt %of the triblock copolymer, and wherein the hydrophobic block associateswith the surface of the core particle and renders the coated particlehydrophilic; and wherein the coated particles have an average size of atleast 5 nm and less than or equal to 1000 nm.
 20. A method of deliveringan anti-microbial agent across a mucosal barrier of a tissue in thegastrointestinal tract, the method comprising delivering to the mucosalbarrier the anti-microbial composition of claim
 19. 21. The method ofclaim 20, wherein the tissue is buccal, esophageal, stomach, smallintestine, large intestine, colon, or rectal tissue.
 22. The method ofclaim 20, wherein the anti-microbial composition is administered byoral, rectal, intraperitoneal, or topical administration.
 23. Amucus-penetrating anti-microbial composition for urogenital delivery,the composition comprising a plurality of coated particles, wherein eachof the coated particles comprise: a core particle comprising ananti-microbial agent, wherein the anti-microbial agent constitutes atleast 80 wt % of the core particle; and a coating comprising asurface-altering agent surrounding the core particle, wherein thesurface-altering agent comprises a triblock copolymer comprising ahydrophilic block—hydrophobic block—hydrophilic block configuration,wherein the hydrophobic block has a molecular weight of at least 2 kDa,and the hydrophilic blocks constitute at least 15 wt % of the triblockcopolymer, and wherein the hydrophobic block associates with the surfaceof the core particle and renders the coated particle hydrophilic; andwherein the coated particles have an average size of at least 5 nm andless than or equal to 1000 nm.
 24. A method of delivering ananti-microbial across a mucosal barrier of a tissue in the urogenitalsystem, the method comprising delivering to the mucosal barrier theanti-microbial composition of claim
 23. 25. The method of claim 24,wherein the anti-microbial composition is administered by vaginal,urethral, or topical administration.
 26. The composition of claim 1,wherein the average size is measured by dynamic light scattering. 27.The composition of claim 1, wherein the core particle is substantiallyfree of a polymeric component.
 28. The composition of claim 1, whereinthe triblock copolymer adsorbed to the core particle is at an averagedensity of at least 0.1 molecules/nm² and less that 1 molecule/nm². 29.The composition of claim 1 wherein the ratio of the total weight of thetriblock copolymer to the total weight of the anti-microbial agentcomprised in the composition is 1:1 to 10:1.
 30. The composition ofclaim 19, wherein the average size is measured by dynamic lightscattering.
 31. The composition of claim 19, wherein the core particleis substantially free of a polymeric component.
 32. The composition ofclaim 19, wherein the triblock copolymer adsorbed to the core particleis at an average density of at least 0.1 molecules/nm² and less that 1molecule/nm².
 33. The composition of claim 19 wherein the ratio of thetotal weight of the triblock copolymer to the total weight of theanti-microbial agent comprised in the composition is 1:1 to 10:1. 34.The composition of claim 23, wherein the average size is measured bydynamic light scattering.
 35. The composition of claim 23, wherein thecore particle is substantially free of a polymeric component.
 36. Thecomposition of claim 23, wherein the triblock copolymer adsorbed to thecore particle is at an average density of at least 0.1 molecules/nm² andless that 1 molecule/nm².
 37. The composition of claim 23 wherein theratio of the total weight of the triblock copolymer to the total weightof the anti-microbial agent comprised in the composition is 1:1 to 10:1.